Method for producing antibody

Abstract

The present invention provides: a method for efficiently producing a recombinant antibody or the like; and a vector and cells used therefor. Specifically, the present invention provides a method for producing an antibody, the method comprising culturing cells containing, in the genome, a total of four coding regions respectively encoding two heavy chains and two light chains of an antibody at a ratio of 1:1:1:1. The present invention also provides a vector containing a total of four coding regions respectively encoding two different heavy chains and two different light chains at a ratio of 1:1:1:1. The vector is useful for efficient production of antibodies in cells. The present invention is particularly suitable for use in the production of multispecific antibodies.

Description

Antibody production method

[0001] The present invention relates to the production of molecules comprising antibodies or antigen-binding fragments thereof.

[0002] In recent years, many biopharmaceuticals, such as antibodies and physiologically active proteins, have been produced. In particular, antibody drugs often require a significant amount of antibody as the active ingredient for a single administration. Technology for efficiently producing recombinant antibodies in animal cells will lead to lower costs for antibody drugs and ensure a stable supply to patients.

[0003] Therefore, there is a need for a method for producing recombinant antibodies with higher production efficiency.

[0004] When preparing host cells for producing recombinant antibodies, a vector containing one copy of DNA encoding the antibody's heavy chain and one copy of DNA encoding the antibody's light chain is typically introduced into the host cells (Non-Patent Documents 1 and 2). The ratio of heavy and light chain genes in this case has also been investigated (Patent Document 1, Non-Patent Document 3, and Non-Patent Document 4). However, in the case of bispecific antibodies in which the antibody has two different heavy chains and two different light chains, the ratio when the four chains contained in the antibody are expressed from a single vector has not been investigated.

[0005] WO2009 / 051108

[0006] Reff ME, et al., Blood. 1994 Jan 15;83(2):435-45.Presta LG, et al., Cancer Res. 1997 Oct 15;57(20):4593-9.Schlatter S, et al., Biotechnol. Prog., 2005 Jan-Feb;21(1): 122-133Koga H, et al. al., MAbs. 2023 Jan-Dec;15(1):2222441

[0007] An objective of the present invention is to provide a method for producing a molecule comprising an antibody or an antigen-binding fragment thereof. Another objective of the present invention is to provide nucleic acids encoding the antibody or molecule, cells harboring the nucleic acids, vectors carrying the nucleic acids, and the like, which are useful for producing a molecule comprising an antibody or an antigen-binding fragment thereof.

[0008] Natural antibodies consist of two heavy chains and two light chains, which pair to form a tetramer. In natural antibodies, the two heavy chains and the two light chains are identical. Therefore, to produce a recombinant antibody, the genes encoding the heavy chain and the light chain are expressed in a host cell, as described above. For example, Schlatter et al. (Schlatter S, et al., Biotechnol. Prog., 2005 Jan-Feb;21(1):122-133; Non-Patent Document 3) have shown that the optimal ratio of H chain genes (hc) to L chain genes (lc) for antibody production is hc:lc = 3:2.

[0009] However, when expressing the four antibody chains from a single vector, it is not known what ratio of coding sequences is optimal for antibody production. In particular, in the case of multispecific antibodies such as bispecific antibodies, the two heavy chains and the two light chains contained in the antibody are generally different from each other, and such antibodies do not exist in nature. Knowledge is limited regarding the optimal ratio of the four chains to be contained in a single vector for such antibodies, making it difficult to predict.

[0010] Therefore, in order to produce an antibody containing two (i.e., two different) heavy chains and two (i.e., two different) light chains, the present inventors investigated antibody production when these four chains were expressed in cells from a single vector.

[0011] The present inventors used FAST-Ig® antibodies, which are bispecific antibodies containing four different chains (two heavy chains and two light chains) and were produced using FAST-Ig® technology, as an example. They expressed the coding regions for each chain from a single vector at various copy ratios, and measured the amount of antibody in the expression product (whole titer) and the proportion of antibody in which the two heavy chains were heteropaired to form the desired bispecific antibody (i.e., heteropaired) (BiAb ​​titer). Here, the coding regions encoding each chain are independent coding regions (i.e., separated coding regions), and each chain expressed from each coding region is expressed as a separate polypeptide chain. As a result, it was found that encoding the copy number ratio of the coding regions for the first heavy and light chains (referred to as H1 and L1, respectively) and the second heavy and light chains (referred to as H2 and L2, respectively) in a single vector at H1:L1:H2:L2 = 1:1:1:1 resulted in significantly better results in terms of both antibody quantity and BiAb ratio compared to expression at other ratios.

[0012] The present inventors have also discovered that antibody molecules composed of H1, L1, H2, and L2 can be produced with high efficiency using expression vectors that contain a total of four coding regions, H1, L1, H2, and L2, in a single vector, particularly expression vectors that contain one of each of these four coding regions (i.e., a total of four coding regions) in a single vector, or expression vectors that contain two of each of these four coding regions (i.e., a total of eight coding regions) in a single vector.

[0013] Thus, the present invention has discovered a method for high antibody production by adjusting the copy number ratio of the four coding regions in an expression system in which four coding regions encoding two heavy chains and two light chains of an antibody are expressed from a single vector.

[0014] Specifically, the present invention relates to a method for producing an antibody comprising two types of heavy chains and two types of light chains, or a polypeptide comprising a fragment thereof, by expressing the coding regions of two types of heavy chains and light chains in a 1:1:1:1:1 ratio. The present invention also provides cells and vectors useful for this purpose, comprising the coding regions of two types of heavy chains and light chains in a 1:1:1:1:1 ratio. Specifically, the present invention provides the following:

[0015] [A1] A recombinant vector comprising, in a single vector, four coding regions encoding two different antibody heavy chains or antigen-binding fragments thereof and two different light chains or antigen-binding fragments thereof corresponding to each heavy chain, in a 1:1:1:1 ratio. [A2] A recombinant vector comprising, in a single vector, equal copies of four coding regions encoding two different antibody heavy chains or antigen-binding fragments thereof and two different light chains or antigen-binding fragments thereof corresponding to each heavy chain. [A3] The vector according to [A1] or [A2], wherein the epitope bound by one of the two different heavy and light chain pairs is the same as the epitope bound by the second of the two different heavy and light chain pairs. [A4] The vector according to any one of [A1] to [A3], wherein the target molecule bound by the first of the two different heavy and light chain pairs is the same as the target molecule bound by the second of the two different heavy and light chain pairs. [A5] The vector according to any one of [A1], [A2], and [A4], wherein the epitope bound by a first heavy chain and light chain pair of the two different heavy chains and light chains is different from the epitope bound by a second heavy chain and light chain pair. [A6] The vector according to any one of [A1], [A2], and [A5], wherein the target molecule bound by a first heavy chain and light chain pair of the two different heavy chains and light chains is different from the target molecule bound by a second heavy chain and light chain pair. [A7] The vector according to any one of [A1], [A2], [A4] to [A6], wherein the antibody is a multispecific antibody. [A8] The vector according to any one of [A1], [A2], [A4] to [A7], wherein the antibody is a bispecific antibody.

[0016] [A9] The vector according to any one of [A1] to [A8], wherein the two different heavy chains are heavy chains that are more prone to heteropairing than homopairing. [A10] The vector according to any one of [A1] to [A9], wherein one or both of the two different heavy chains have a mutation that promotes heteropairing and / or suppresses homopairing. [A11] The vector according to any one of [A1] to [A10], wherein at least one of the two different heavy chains has a mutation that suppresses homopairing of that chain due to steric hindrance. [A12] The vector according to any one of [A1] to [A11], wherein pairing between one heavy chain of the antibody and / or pairing between the other heavy chains is suppressed by steric hindrance due to knob-into-hole and / or charge repulsion of charged amino acids. [A13] The vector according to any one of [A1] to [A12], wherein knob-into-hole is introduced into the two different heavy chains. [A14] The vector according to any one of [A1] to [A13], wherein the amino acid at EU numbering position 366 (EU numbering) of one of the two different heavy chains is Trp (W) and / or the amino acid at EU numbering position 349 is Cys (C), and the amino acid at EU numbering position 356 (EU numbering) of the other heavy chain is Cys (C), the amino acid at EU numbering position 366 is Ser (S), the amino acid at EU numbering position 368 is Ala (A), and / or the amino acid at EU numbering position 407 is Val (V). [A15] The vector according to [A14], wherein the amino acid at EU numbering position 366 (EU numbering) of one of the two different heavy chains is Trp (W), and the amino acid at EU numbering position 407 of the other heavy chain is Val (V). [A16] The vector according to any one of [A1] to [A15], wherein the amino acid at EU numbering position 366 is Trp (W) and the amino acid at EU numbering position 349 is Cys (C) in one of the two different heavy chains, and the amino acid at EU numbering position 366 is Ser (S), the amino acid at EU numbering position 368 is Ala (A), and the amino acid at EU numbering position 407 is Val (V) in the other heavy chain. [A17] The vector according to [A16], wherein the amino acid at EU numbering position 356 is also Cys (C) in the other heavy chain.[A18] The vector according to any one of [A1] to [A17], wherein the two different heavy chains are cross-linked by a disulfide bond in the CH3 region. [A19] The vector according to any one of [A1] to [A18], wherein the amino acid at position 354 (EU numbering) of one of the two different heavy chains is Cys(C), and the amino acid at position 349 (EU numbering) of the other heavy chain is Cys(C). [A20] The vector according to any one of [A1] to [A19], wherein at least one of the two different heavy chains has a mutation that suppresses homopairing of that chain due to charge repulsion. [A21] The vector according to any one of [A1] to [A20], wherein at least one of the two different heavy chains has a charged amino acid that suppresses homopairing of that chain.

[0017] [A22] The vector according to any one of [A1] to [A21], wherein a positively or negatively charged amino acid introduced into one of the two different heavy chains repels a charged amino acid of the same charge that is present in or introduced into the same chain, thereby suppressing homopairing of the heavy chains. [A23] The vector according to any one of [A1] to [A22], wherein a positively or negatively charged amino acid is introduced into one of the two different heavy chains, and a charged amino acid of the opposite charge is present in or introduced into the other chain. [A24] The vector according to any one of [A1] to [A23], wherein heteropairing of heavy chains is promoted by an attraction between a positively or negatively charged amino acid introduced into one of the two different heavy chains and a charged amino acid of the opposite charge that is present in or introduced into the other chain. [A25] The vector according to any one of [A1] to [A24], wherein the amino acid at EU numbering position a and the amino acid at EU numbering position b in one of the two different heavy chains have charged amino acids of the same type, and positions a and b are selected from the following combinations (i) to (iii): (i) Position a: EU numbering position 356, Position b: EU numbering position 439; (ii) Position a: EU numbering position 357, Position b: EU numbering position 370; (iii) Position a: EU numbering position 399, Position b: EU numbering position 409. [A26] The vector of any of [A1] to [A25], wherein the amino acid at EU numbering position a in one of the two different heavy chains and the amino acid at EU numbering position b in the other heavy chain have charged amino acids of the same type, and the charge of the charged amino acid in one heavy chain is opposite to the charge of the charged amino acid in the other heavy chain, and positions a and b are selected from the following combinations (i) to (iii):(i) Position a: EU numbering position 356, Position b: EU numbering position 439, (ii) Position a: EU numbering position 357, Position b: EU numbering position 370, (iii) Position a: EU numbering position 399, Position b: EU numbering position 409 [A27] The vector according to any of [A21] to [A26], wherein the charged amino acid is (a) selected from the group consisting of lysine (K), arginine (R), and histidine (H), or (b) an amino acid selected from the group consisting of glutamic acid (E) and aspartic acid (D), and the charge of the amino acid in (a) is opposite to the charge of the amino acid in (b). [A28] The vector according to any of [A1] to [A27], wherein pairing of at least one of the two different heavy chains with its corresponding light chain is promoted compared to pairing with an uncorresponding light chain. [A29] The vector according to any one of [A1] to [A28], wherein pairing of at least one of the two different heavy chains with its corresponding light chain is promoted compared to pairing with an uncorresponding light chain due to attraction between positively charged amino acids and negatively charged amino acids. [A30] The vector according to any one of [A1] to [A29], wherein pairing of at least one of the two different heavy chains with its uncorresponding light chain is suppressed compared to pairing with the corresponding light chain due to repulsion of charged amino acids. [A31] The vector according to any one of [A1] to [A30], wherein pairing of one of the two different heavy chains with the light chain corresponding to the other heavy chain, and pairing of the other heavy chain with the light chain corresponding to the one heavy chain, are suppressed due to charge repulsion of charged amino acids. [A32] The vector according to any one of [A1] to [A31], wherein at least one of the two different heavy chains and two different light chains has a mutation that promotes pairing of the corresponding heavy chain / light chain and / or suppresses pairing of non-corresponding heavy chain / light chain. [A33] The vector according to any one of [A1] to [A32], wherein at least one of the two different heavy chains and either or both of the light chain corresponding to that heavy chain have a mutation that promotes pairing of the heavy chain and light chain.[A34] The vector according to any one of [A1] to [A33], wherein at least one of the two different heavy chains and either or both of the light chains that do not correspond to that heavy chain have a mutation that suppresses pairing between the heavy chain and the light chain. [A35] The vector according to any one of [A1] to [A34], wherein all of the two different heavy chains and the two different light chains have a mutation that promotes pairing between corresponding heavy chains and light chains and / or suppresses pairing between non-corresponding heavy chains and light chains. [A36] The vector according to any one of [A32] to [A35], wherein the mutation at least includes the introduction of a charged amino acid. [A37] The vector according to any one of [A32] to [A36], wherein a positively or negatively charged amino acid has been introduced into at least one of the two different heavy chains, and a charged amino acid of the opposite charge has been introduced into the light chain corresponding to that heavy chain, and / or a charged amino acid of the same charge has been introduced into the non-corresponding light chain. [A38] The vector according to [A37], wherein pairing between the heavy chain and the corresponding light chain is promoted and / or pairing between the heavy chain and the corresponding light chain is suppressed due to attraction between the positively or negatively charged amino acid in the heavy chain into which the amino acid has been introduced and an oppositely charged amino acid in the corresponding light chain, and / or repulsion between the same type of charged amino acid in the non-corresponding light chain.

[0018] [A39] The vector according to any one of [A1] to [A38], wherein one of the two different heavy chains has a charged amino acid in CH1, and the light chain corresponding to the other heavy chain has a charged amino acid of the same charge in CL. [A40] The vector according to [A39], wherein pairing of one of the heavy chains with the light chain is inhibited by charge repulsion. [A41] The vector according to any one of [A1] to [A40], wherein one of the two different heavy chains has a charged amino acid in CH1, and the light chain corresponding to the other heavy chain has a charged amino acid of the same charge as the charged amino acid of the opposite charge in CH1, and the light chain corresponding to the one heavy chain has a charged amino acid of the same charge in CL. [A42] The vector according to [A41], wherein pairing between one heavy chain and the light chain corresponding to the other heavy chain is inhibited by charge repulsion, thereby inhibiting pairing between the other heavy chain and the light chain corresponding to the one heavy chain. [A43] The vector according to any of [A1] to [A42], wherein the amino acid at position 147 and / or 175 (EU numbering) in CH1 of one of the two different heavy chains and any of the amino acids at positions 131, 160, and 180 (EU numbering) in CL of the light chain corresponding to the other heavy chain, or any combination thereof, are mutually electrically repulsive. [A44] The vector according to any of [A1] to [A43], wherein the amino acid at position 213 (EU numbering) in CH1 of one of the two different heavy chains and the amino acid at position 123 (EU numbering) in CL of the light chain corresponding to the other heavy chain, are mutually electrically repulsive. [A45] The vector according to any one of [A1] to [A44], wherein either or both of the amino acid residues at positions 147 and 175 (EU numbering) in CH1 of one of the two different heavy chains and either or any combination of the amino acids at positions 131, 160, and 180 (EU numbering) in CL of the light chain corresponding to the other heavy chain are electrically repulsive amino acids.[A46] The vector according to any one of [A1] to [A45], wherein an amino acid residue contained in CH1 of one of the two different heavy chains and an amino acid residue contained in CL of the light chain corresponding to the other heavy chain are amino acids that are electrically repulsive to each other, and these amino acid residues are selected from any one of the following (a) to (j): (a) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residue at position 160 in CL according to Kabat numbering; (b) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residue at position 131 in CL according to Kabat numbering; (c) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residue at position 180 in CL according to Kabat numbering; (d) the amino acid residue at position 147 in CH1 according to EU numbering and the amino acid residue at position 131 in CL according to Kabat numbering; (e) the amino acid residue at position 147 in CH1 according to EU numbering and the amino acid residue at position 160 in CL according to Kabat numbering; (f) (g) the amino acid residue at position 213 in CH1 according to EU numbering and the amino acid residue at position 123 in CL according to Kabat numbering; (h) the amino acid residues at positions 147 and 175 in CH1 according to EU numbering and the amino acid residues at positions 131 and 160 in CL according to Kabat numbering; (i) the amino acid residues at positions 147 and 175 in CH1 according to EU numbering and the amino acid residues at positions 131 and 180 in CL according to Kabat numbering; and (j) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residues at positions 131 and 160 in CL according to Kabat numbering.[A47] The vector according to any one of [A43] to [A46], wherein the electrically repulsive amino acid is (a) selected from the group consisting of lysine (K), arginine (R), and histidine (H), or (b) selected from the group consisting of glutamic acid (E) and aspartic acid (D). [A48] The vector according to any one of [A1] to [A47], wherein the antibody is a FAST-Ig (registered trademark) antibody.

[0019] [A49] The vector according to any one of [A1] to [A48], wherein the isoelectric point of one of the two different heavy chains differs from that of the other. [A50] The vector according to [A49], wherein the isoelectric point of one of the two different heavy chains differs from that of the other by 0.5 or more. [A51] The vector according to any one of [A1] to [A50], into which a mutation has been introduced that increases the difference in isoelectric point between one of the two different heavy chains and the other heavy chain. [A52] In one of the two different heavy chains, (i) amino acid residues at positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 97, 105, 108, 110, and 112 (Kabat numbering) in the variable region of the heavy chain, and (ii) at least one amino acid residue selected from amino acid residues at positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 (EU numbering) in the heavy chain constant region is charged; and in the other heavy chain, (i) (ii) amino acid residues at positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 97, 105, 108, 110, and 112 according to the Kabat numbering system in the heavy chain variable region; and The vector of any of [A1] to [A51], wherein at least one amino acid residue selected from amino acid residues at positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 (EU numbering) in the heavy chain constant region has an opposite charge to that of the charged amino acid residue selected in the other heavy chain, or is uncharged.[A53] The vector according to [A52], wherein the combination of the charged amino acid residue and the amino acid residue having the opposite charge to that of the charged amino acid residue is selected from amino acid residues included in either the following group (a) or (b): (a) glutamic acid (E), aspartic acid (D); (b) lysine (K), arginine (R), histidine (H). [A54] The vector according to any of [A1] to [A53], wherein one heavy chain and the other heavy chain of the two different heavy chains are derived from IgG1 and IgG4, respectively, or IgG1 and IgG2, respectively.

[0020] [A55] The vector according to any one of [A1] to [A54], wherein the antibody binds to a complex formed by HLA-DQ2.5 and a gluten peptide. [A56] The vector according to [A55], wherein the antibody specifically binds to a complex formed by HLA-DQ2.5 and a gluten peptide. [A57] The vector according to [A55] or [A56], wherein the antibody binds to a complex formed by HLA-DQ2.5 and a first gluten peptide and a complex formed by HLA-DQ2.5 and a second gluten peptide. [A58] The vector according to any one of [A55] to [A57], wherein the antibody has substantially no binding activity to either or both of HLA-DQ2.5-positive PBMC B cells and Ba / F3 cells expressing HLA-DQ2.5. [A59] The vector according to any one of [A55] to [A58], wherein the gluten peptide is an immunodominant peptide associated with celiac disease. [A60] The vector according to any one of [A55] to [A59], wherein the gluten peptide is selected from the group consisting of a 33-mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4a gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, a hordein 1 peptide, a hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, and a 26-mer gliadin peptide, or an ω2 gliadin peptide, a BC hordein peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, a γ4a gliadin peptide, and a γ4d gliadin peptide. [A61] The vector according to any one of [A55] to [A60], wherein the antibody has substantially no binding activity to HLA-DQ2.5 in the form of a complex with a peptide unrelated to gluten peptides. [A62] The peptide unrelated to gluten peptides is a CLIP peptide, a Hepatitis B virus 1 peptide, a Salmonella peptide, a Mycobacterium bovis peptide, or a peptide unrelated to gluten peptides.[A61] The vector according to [A61], wherein the antibody is at least one peptide selected from the group consisting of HLA-DQ2.5 (HLA-DQ2.5) peptide, thyroxidase peptide, and thyroxidase peptide. [A63] The vector according to any one of [A55] to [A62], wherein the antibody is a humanized antibody. [A64] The vector according to any one of [A55] to [A63], wherein the amino acid sequence of the antibody has been modified so that the binding activity to a complex formed by HLA-DQ2.5 and a gluten peptide is enhanced. [A65] The vector according to [A64], wherein the gluten peptide contained in the complex is selected from the group consisting of ω2 gliadin peptide, BC hordein peptide, γ1 gliadin peptide, γ2 gliadin peptide, γ4a gliadin peptide, and γ4d gliadin peptide. [A66] The vector according to any one of [A55] to [A65], wherein one, two, three, or all of the sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in the following (a) to (d) in the heavy chain and light chain of the antibody have been modified to amino acid residues that are electrostatically repulsive to each other: (a) the amino acid residue in the heavy chain constant region (CH1) that is at position 175 according to EU numbering and the amino acid residue in the light chain constant region (CL) that is at position 131 according to Kabat numbering, (b) the amino acid residue in CH1 that is at position 175 according to EU numbering and the amino acid residue in CL that is at position 160 according to Kabat numbering, (c) the amino acid residue in CH1 that is at position 175 according to EU numbering and the amino acid residues in CL that are at positions 131 and 160 according to Kabat numbering, (d) The amino acid residues in CH1 are at positions 147 and 175 according to EU numbering, and the amino acid residues in CL are at positions 131 and 160 according to Kabat numbering. [A67] The vector of any of [A55] to [A66], wherein two or more amino acid residues forming an interface between the heavy chain variable region and the light chain variable region of the antibody are amino acid residues that electrostatically repel each other.[A68] The vector of [A67], wherein the mutually electrostatically repulsive amino acid residues are one or two sets of amino acid residues selected from the group consisting of the following sets of amino acid residues (a) and (b): (a) an amino acid residue in the heavy chain variable region that is at position 39 according to the Kabat numbering, and an amino acid residue in the light chain variable region that is at position 38 according to the Kabat numbering, (b) an amino acid residue in the heavy chain variable region that is at position 45 according to the Kabat numbering, and an amino acid residue in the light chain variable region that is at position 44 according to the Kabat numbering. [A69] The vector of any of [A66] to [A68], wherein the mutually electrostatically repulsive amino acid residues are selected from amino acid residues included in either set (X) or (Y) below: (X) glutamic acid (E), aspartic acid (D), (Y) lysine (K), arginine (R), histidine (H). [A70] The vector of any of [A55] to [A69], wherein the antibody comprises an Fc domain. [A71] The vector of any of [A55] to [A70], wherein the antibody comprises an Fc domain that exhibits reduced binding affinity for human Fcγ receptors compared to a native human IgG1 Fc domain. [A72] The vector of [A70] or [A71], wherein the Fc domain comprises Arg at positions 235 and 236 (EU numbering). [A73] The vector of any of [A70] to [A72], wherein the Fc domain comprises the following (e1) or (e2): (e1) a first Fc region subunit comprising Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407 (EU numbering), and a second Fc region subunit comprising Cys at position 354 and Trp at position 366; (e2) a first Fc region subunit comprising Glu at position 439 (EU numbering), and a second Fc region subunit comprising Lys at position 356. [A74] The vector of any of [A70] to [A73], wherein the Fc domain exhibits stronger FcRn-binding affinity for human FcRn compared to native human IgG1 Fc domain.[A75] The vector of any of [A70] to [A74], wherein the first and / or second Fc region subunit of the Fc domain comprises, according to EU numbering, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440. [A76] The vector of any of [A70] to [A74], wherein the first and / or second Fc region subunit of the Fc domain comprises, according to EU numbering, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440. [A77] The vector of any one of [A55] to [A76], wherein the antibody comprises one or more of the following amino acid residues (i) to (xii): (i) a glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region; (ii) a glutamic acid at position 147 (EU numbering) in the heavy chain constant region; (iii) a glutamic acid or lysine at position 131 (Kabat numbering) in the light chain constant region; (iv) a glutamic acid or lysine at position 160 (Kabat numbering) in the light chain constant region; (v) an arginine at position 235 (EU numbering) in the heavy chain constant region; (vi) an arginine at position 236 (EU numbering) in the heavy chain constant region; (vii) a lysine at position 356 (EU numbering) in the heavy chain constant region; (viii) (ix) alanine at position 434 (EU numbering) in the heavy chain constant region; (x) arginine at position 438 (EU numbering) in the heavy chain constant region; (xi) glutamic acid at position 439 (EU numbering) in the heavy chain constant region; (xii) glutamic acid at position 440 (EU numbering) in the heavy chain constant region. [A78] The vector of any one of [A55] to [A77], wherein the antibody is a bispecific antibody comprising: a first heavy chain comprising lysine at position 175 (EU numbering), arginine at position 235 (EU numbering), arginine at position 236 (EU numbering), leucine at position 428 (EU numbering), alanine at position 434 (EU numbering), arginine at position 438 (EU numbering), glutamic acid at position 439 (EU numbering), and glutamic acid at position 440 (EU numbering);a first light chain comprising a glutamic acid at position 131 (Kabat numbering) and a glutamic acid at position 160 (Kabat numbering); a second heavy chain comprising a glutamic acid at position 147 (EU numbering), a glutamic acid at position 175 (EU numbering), an arginine at position 235 (EU numbering), an arginine at position 236 (EU numbering), a lysine at position 356 (EU numbering), a leucine at position 428 (EU numbering), an alanine at position 434 (EU numbering), an arginine at position 438 (EU numbering), and a glutamic acid at position 440 (EU numbering); and a second light chain comprising a lysine at position 131 (Kabat numbering) and a lysine at position 160 (Kabat numbering). [A79] The vector of any of [A55] to [A78], wherein the antibody's first heavy chain further comprises a glutamic acid at position 419 (EU numbering), a proline at position 445 (EU numbering), and amino acid deletions at positions 446 and 447 (EU numbering); and the second heavy chain further comprises a lysine at position 196 (EU numbering), a proline at position 445 (EU numbering), and amino acid deletions at positions 446 and 447 (EU numbering). [A80] the antibody, wherein the first heavy chain further contains a glycine at position 16 (Kabat numbering), an alanine at position 32 (Kabat numbering), a lysine at position 61 (Kabat numbering), a valine at position 35a (Kabat numbering), an alanine at position 50 (Kabat numbering), a glutamic acid at position 64 (Kabat numbering), a threonine at position 73 (Kabat numbering), a glutamic acid at position 95 (Kabat numbering), and a valine at position 102 (Kabat numbering); the first light chain further comprises a glutamic acid at position 28 (Kabat numbering), a tyrosine at position 55 (Kabat numbering), a glutamic acid or tyrosine at position 56 (Kabat numbering), a glutamic acid at position 92 (Kabat numbering), a valine at position 94 (Kabat numbering), and an alanine at position 95a (Kabat numbering);the second heavy chain contains a glutamic acid at position 28 (Kabat numbering), an alanine or glutamic acid at position 30 (Kabat numbering), a glutamic acid at position 31 (Kabat numbering), a tryptophan at position 32 (Kabat numbering), a phenylalanine at position 34 (Kabat numbering), a methionine at position 35 (Kabat numbering), a serine at position 35a (Kabat numbering), a serine at position 50 (Kabat numbering), a glutamic acid or glycine at position 61 (Kabat numbering), The vector of any of [A55] to [A79], further comprising a glutamic acid at position 64 (Kabat numbering), and a glutamic acid at position 65 (Kabat numbering); and the second light chain further comprising a threonine at position 25 (Kabat numbering), a lysine at position 54 (Kabat numbering), a glutamic acid at position 56 (Kabat numbering), a leucine at position 67 (Kabat numbering), a glutamine at position 79 (Kabat numbering), and a lysine at position 94 (Kabat numbering). [A81] The vector according to any one of [A55] to [A80], wherein the antibody is a bispecific antibody described in WO2022 / 059766, and is any one of the following bispecific antibodies: (1) DQN0344H0976 / L0591 / / DQN0385H1270 / L0722-F6 (a bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1270 / L0722) (2) DQN0344H0976 / L0591 / / DQN0385H1270 / L0681-F6 (a bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1270 / L0681) (3) DQN0344H0976 / L0591 / / DQN0385H1352 / L0681-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1352 / L0681) (4) DQN0344H0976 / L0591 / / DQN0385H1527 / L0605-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1527 / L0605) (5)DQN0344H0976 / L0591 / / DQN0385H1255 / L0605-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1255 / L0605) (6) DQN0344H1013 / L0620 / / DQN0385H1270 / L0722-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1270 / L0722) (7) DQN0344H1013 / L0620 / / DQN0385H1521 / L0605-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1521 / L0605) (8) DQN0344H1013 / L0620 / / DQN0385H1270 / L0681-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1270 / L0681) (9) DQN0344H1013 / L0620 / / DQN0385H1352 / L0681-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1352 / L0681) (10) DQN0344H1013 / L0620 / / DQN0385H1353 / L0681-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1353 / L0681) (11) DQN0344H0976 / L0591 / / DQN0385H1521 / L0605-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1521 / L0605) (12) DQN0344H0976 / L0591 / / DQN0385H1353 / L0681-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1353 / L0681) (13) DQN0344H1013 / L0620 / / DQN0385H1255 / L0605-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1255 / L0605) (14) DQN0344H1013 / L0620 / / DQN0385H1527 / L0605-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1527 / L0605) (15)A first polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the first heavy chain sequence of the bispecific antibody of any one of (1) to (14); a second polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the first light chain sequence of the bispecific antibody of any one of (1) to (14); any one of (1) to (14). a third polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the second heavy chain sequence of the bispecific antibody of (1); and a fourth polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the second light chain sequence of the bispecific antibody of any one of (1) to (14). [A82] The vector according to any one of [A55] to [A81], wherein the antibody is a bispecific antibody comprising a combination of four polypeptide chains selected from the group consisting of the following (1) to (15): (1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, as well as a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second light chain comprising the amino acid sequence of SEQ ID NO: 6; (2) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, as well as a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (3) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, as well as a second heavy chain comprising the amino acid sequence of SEQ ID NO: 8 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (4) (5) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10;(6) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second light chain comprising the amino acid sequence of SEQ ID NO: 6; (7) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; (8) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (9) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 8 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (10) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (11) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; (12) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (13) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; (14)a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; and (15) A first polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a first heavy chain sequence described in any one of (1) to (14); a second polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a first light chain sequence described in any one of (1) to (14); a third polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a second heavy chain sequence described in any one of (1) to (14); and a fourth polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a second light chain sequence described in any one of (1) to (14). [A83] The vector according to any one of [A55] to [A82], wherein the antibody is a bispecific antibody comprising a combination of four polypeptide chains selected from the group consisting of the following (1) to (2): (1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a first light chain comprising the amino acid sequence of SEQ ID NO: 15, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a second light chain comprising the amino acid sequence of SEQ ID NO: 17, or (2) a first polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the first heavy chain sequence described in (1); and a second polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the first light chain sequence described in (1); a third polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the second heavy chain sequence set forth in (1); anda fourth polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the second light chain sequence set forth in

[0021] [A84] The vector according to any one of [A1] to [A83], comprising two or more copies of each of the four coding regions. [A85] The vector according to any one of [A1] to [A83], comprising one copy of each of the four coding regions. [A86] The vector according to any one of [A1] to [A84], comprising two copies of each of the four coding regions. [A87] The vector according to any one of [A1] to [A86], wherein the four coding regions are transcribed as different mRNA molecules. [A88] The vector according to any one of [A1] to [A87], wherein the four coding regions are transcribed as independent transcription units. [A89] The vector according to any one of [A1] to [A88], wherein the sense strands of the four coding regions are encoded on the same strand within the vector. [A90] The vector according to any one of [A1] to [A89], wherein the four coding regions are transcribed from independent expression units. [A91] The vector according to [A90], wherein the expression unit comprises a promoter, a coding sequence, and a poly(A) addition signal. [A92] The vector according to any one of [A1] to [A92], wherein a promoter is linked to each of the four coding regions. [A93] The vector according to [A92], wherein the promoters linked to each of the four coding regions are the same promoter. [A94] The vector according to any one of [A91] to [A93], wherein the promoter is a pol II promoter. [A95] The vector according to any one of [A1] to [A94], wherein the vector is a prokaryotic or eukaryotic expression vector. [A96] The vector according to any one of [A1] to [A94], wherein the vector is a eukaryotic expression vector. [A97] The vector according to [A96], wherein the vector is an animal expression vector. [A98] The vector according to [A96] or [A97], wherein the vector is a mammalian expression vector. [A99] The vector according to any one of [A91] to [A98], wherein the promoter is selected from the group consisting of a CMV promoter, a CAG promoter, an EF1a promoter, an RSV promoter, and an SV40 promoter. [A100] The vector according to any one of [A91] to [A99], wherein the promoter is an actin promoter.[A101] The vector according to [A100], wherein the promoter is a β-actin promoter. [A102] The vector according to [A101], wherein the promoter is a chicken β-actin promoter. [A103] The vector according to any one of [A91] to [A102], wherein the promoter further comprises an enhancer. [A104] The vector according to [A103], wherein the enhancer is a CMV E1 enhancer. [A105] The vector according to any one of [A91] to [A104], wherein the promoter is a CAG promoter.

[0022] [B1] A cell into which the vector according to any one of [A1] to [A105] has been introduced. [B2] The cell according to [B1], into which one copy of the vector has been introduced. [B3] The cell according to [B1], into which multiple copies of the vector have been introduced. [B4] The cell according to [B1], into which two or more copies of the vector have been introduced. [B5] The cell according to [B1], into which three or more copies of the vector have been introduced. [B6] The cell according to any one of [B1] to [B5], into which the vector has been transiently introduced. [B7] The cell according to any one of [B1] to [B6], into which the vector has been episomally introduced. [B8] The cell according to any one of [B1] to [B5], into which the vector has been stably introduced. [B9] The cell according to any one of [B1] to [B5] or [B8], into which the vector has been introduced into the genome of the cell. [B10] The cell according to [B9], wherein the vector is introduced into a hotspot in the cell's genome.

[0023] [B11] A cell comprising four exogenous nucleic acids in a 1:1:1:1 ratio, each encoding two different antibody heavy chains or antigen-binding fragments thereof and two different light chains or antigen-binding fragments thereof corresponding to each heavy chain. [B12] A cell comprising equal copies of four coding regions, each encoding two different antibody heavy chains or antigen-binding fragments thereof and two different light chains or antigen-binding fragments thereof corresponding to each heavy chain. [B13] The cell according to any of [B11] to [B12], wherein the epitope bound by a first pair of heavy and light chains among the two different heavy and light chains is the same as the epitope bound by a second pair of heavy and light chains. [B14] The cell according to any of [B11] to [B13], wherein the target molecule bound by a first pair of heavy and light chains among the two different heavy and light chains is the same as the target molecule bound by a second pair of heavy and light chains. [B15] The cell according to any one of [B11], [B12], or [B14], wherein the epitope bound by a first pair of heavy chains and light chains is different from the epitope bound by a second pair of heavy chains and light chains. [B16] The cell according to any one of [B11], [B12], or [B15], wherein the target molecule bound by a first pair of heavy chains and light chains is different from the target molecule bound by a second pair of heavy chains and light chains.

[0024] [B17] The cell according to any one of [B11], [B12], [B14] to [B16], wherein the antibody is a multispecific antibody. [B18] The cell according to any one of [B11], [B12], [B14] to [B17], wherein the antibody is a bispecific antibody.

[0025] [B19] The cell according to any one of [B11] to [B18], wherein the two different heavy chains are heavy chains that are more prone to heteropairing than homopairing. [B20] The cell according to any one of [B11] to [B19], wherein one or both of the two different heavy chains have a mutation that promotes heteropairing and / or suppresses homopairing. [B21] The cell according to any one of [B11] to [B20], wherein at least one of the two different heavy chains has a mutation that suppresses homopairing of that chain due to steric hindrance. [B22] The cell according to any one of [B11] to [B21], wherein pairing between one heavy chain of the antibody and / or pairing between the other heavy chains is suppressed by steric hindrance due to knob-into-hole and / or charge repulsion of charged amino acids. [B23] The cell according to any one of [B11] to [B22], wherein the two different heavy chains have a knob-into-hole introduced therein. [B24] The cell according to any one of [B11] to [B23], wherein the amino acid at position 366 (EU numbering) of one heavy chain is Trp (W) and / or the amino acid at position 349 (EU numbering) is Cys (C), and the amino acid at position 356 (EU numbering) of the other heavy chain is Cys (C), the amino acid at position 366 (EU numbering) is Ser (S), the amino acid at position 368 (EU numbering) is Ala (A), and / or the amino acid at position 407 (EU numbering) is Val (V). [B25] The cell according to [B24], wherein the amino acid at EU numbering position 366 in one of the two different heavy chains is Trp (W), and the amino acid at EU numbering position 407 in the other heavy chain is Val (V). [B26] The cell according to any one of [B11] to [B25], wherein the amino acid at EU numbering position 366 in one of the two different heavy chains is Trp (W) and the amino acid at EU numbering position 349 in one of the two different heavy chains is Cys (C), and the amino acid at EU numbering position 366 in the other heavy chain is Ser (S), the amino acid at EU numbering position 368 is Ala (A), and the amino acid at EU numbering position 407 is Val (V). [B27] The cell according to [B26], wherein the amino acid at EU numbering position 356 in the other heavy chain is also Cys (C).[B28] The cell according to any one of [B11] to [B27], wherein the two different heavy chains are cross-linked by a disulfide bond in the CH3 region. [B29] The cell according to any one of [B11] to [B28], wherein the amino acid at position 354 (EU numbering) of one of the two different heavy chains is Cys(C), and the amino acid at position 349 (EU numbering) of the other heavy chain is Cys(C). [B30] The cell according to any one of [B11] to [B29], wherein at least one of the two different heavy chains has a mutation that prevents homopairing of that chain due to charge repulsion. [B31] The cell according to any one of [B11] to [B30], wherein at least one of the two different heavy chains has a charged amino acid that prevents homopairing of that chain. [B32] The cell according to any one of [B11] to [B31], wherein homopairing of the heavy chains is suppressed by mutual repulsion between a positively or negatively charged amino acid introduced into one of the two different heavy chains and a charged amino acid of the same charge present or introduced in the same chain. [B33] The cell according to any one of [B11] to [B32], wherein a positively or negatively charged amino acid has been introduced into one of the two different heavy chains, and a charged amino acid of the opposite charge is present or introduced in the other chain. [B34] The cell according to any one of [B11] to [B33], wherein heteropairing of heavy chains is promoted by mutual attraction between a positively or negatively charged amino acid introduced into one of the two different heavy chains and a charged amino acid of the opposite charge present or introduced in the other chain. [B35] The cell according to any one of [B11] to [B34], wherein the amino acid at EU numbering position a and the amino acid at EU numbering position b of one of the two different heavy chains have charged amino acids of the same type, and positions a and b are selected from the following combinations (i) to (iii):(i) Position a: EU numbering position 356, Position b: EU numbering position 439; (ii) Position a: EU numbering position 357, Position b: EU numbering position 370; (iii) Position a: EU numbering position 399, Position b: EU numbering position 409. [B36] The cell according to any of [B11] to [B35], wherein the amino acid at EU numbering position a in one of the two different heavy chains and the amino acid at EU numbering position b in the other heavy chain have charged amino acids of the same type, and the charge of the charged amino acid in one heavy chain is opposite to the charge of the charged amino acid in the other heavy chain, and positions a and b are selected from the following combinations (i) to (iii): (i) Position a: EU numbering position 356, Position b: EU numbering position 439 (ii) Position a: EU numbering position 357, Position b: EU numbering position 370 (iii) Position a: EU numbering position 399, Position b: EU numbering position 409 [B37] A cell described in any of [B31] to [B36], wherein the charged amino acid is (a) selected from the group consisting of lysine (K), arginine (R), and histidine (H), or (b) an amino acid selected from the group consisting of glutamic acid (E) and aspartic acid (D), and the charge of the amino acid in (a) is opposite to the charge of the amino acid in (b).

[0026] [B38] The cell according to any one of [B11] to [B37], wherein pairing of at least one of the two different heavy chains with its corresponding light chain is promoted compared to pairing with an unmatched light chain. [B39] The cell according to any one of [B11] to [B38], wherein pairing of at least one of the two different heavy chains with its corresponding light chain is promoted compared to pairing with an unmatched light chain due to attraction between positively charged amino acids and negatively charged amino acids. [B40] The cell according to any one of [B11] to [B39], wherein pairing of at least one of the two different heavy chains with its unmatched light chain is suppressed compared to pairing with the corresponding light chain due to repulsion of charged amino acids. [B41] The cell according to any one of [B11] to [B40], wherein pairing of one of the two different heavy chains with the light chain corresponding to the other heavy chain, and pairing of the other heavy chain with the light chain corresponding to the one heavy chain, are inhibited by charge repulsion of charged amino acids. [B42] The cell according to any one of [B11] to [B41], wherein at least one of the two different heavy chains and two different light chains has a mutation that promotes pairing of corresponding heavy chains / light chains and / or inhibits pairing of non-corresponding heavy chains / light chains. [B43] The cell according to any one of [B11] to [B42], wherein at least one of the two different heavy chains and either or both of the light chain corresponding to that heavy chain have a mutation that promotes pairing of the heavy chain and light chain. [B44] The cell according to any one of [B11] to [B43], wherein at least one of the two different heavy chains and either or both of the light chains that do not correspond to that heavy chain have a mutation that suppresses pairing between the heavy chain and the light chain. [B45] The cell according to any one of [B11] to [B44], wherein all of the two different heavy chains and the two different light chains have a mutation that promotes pairing between corresponding heavy chains and light chains and / or suppresses pairing between non-corresponding heavy chains and light chains. [B46] The cell according to any one of [B42] to [B45], wherein the mutation at least includes the introduction of a charged amino acid.[B47] The cell according to any one of [B42] to [B46], wherein a positively or negatively charged amino acid has been introduced into at least one of the two different heavy chains, and a charged amino acid of the opposite charge has been introduced into the light chain corresponding to the heavy chain, and / or a charged amino acid of the same charge has been introduced into the non-corresponding light chain. [B48] The cell according to [B47], wherein pairing of the heavy chain with the corresponding light chain is promoted and / or inhibited due to attraction between the charged amino acid of the heavy chain into which the positively or negatively charged amino acid has been introduced and an oppositely charged amino acid introduced into the corresponding light chain, and / or repulsion between the charged amino acid of the same charge introduced into the non-corresponding light chain.

[0027] [B49] The cell according to any one of [B11] to [B48], wherein one of the two different heavy chains has a charged amino acid in CH1, and the light chain corresponding to the other heavy chain has a charged amino acid of the same charge in CL. [B50] The cell according to [B49], wherein pairing of one of the heavy chains with the light chain is inhibited by charge repulsion. [B51] The cell according to any one of [B11] to [B50], wherein one of the two different heavy chains has a charged amino acid in CH1, the light chain corresponding to the other heavy chain has a charged amino acid of the same charge as the charged amino acid of the opposite charge in CH1, and the light chain corresponding to the one heavy chain has a charged amino acid of the same charge as the charged amino acid of the opposite charge in CL. [B52] The cell according to [B51], wherein pairing between one heavy chain and the light chain corresponding to the other heavy chain is inhibited by charge repulsion, and pairing between the other heavy chain and the light chain corresponding to the one heavy chain is inhibited. [B53] The cell according to any of [B11] to [B52], wherein the amino acid at position 147 and / or 175 (EU numbering) in CH1 of one of the two different heavy chains and any of the amino acids at positions 131, 160, and 180 (EU numbering) in CL of the light chain corresponding to the other heavy chain, or any combination thereof, are mutually electrically repulsive. [B54] The cell according to any of [B11] to [B53], wherein the amino acid at position 213 (EU numbering) in CH1 of one of the two different heavy chains and the amino acid at position 123 (EU numbering) in CL of the light chain corresponding to the other heavy chain are mutually electrically repulsive. [B55] A cell according to any one of [B11] to [B54], wherein either or both of the amino acid residues at positions 147 and 175 (EU numbering) in CH1 of one of the two different heavy chains and either or any combination of the amino acids at positions 131, 160, and 180 (EU numbering) in CL of the light chain corresponding to the other heavy chain are electrically repulsive amino acids.[B56] A cell according to any one of [B11] to [B55], wherein an amino acid residue contained in CH1 of one of the two different heavy chains and an amino acid residue contained in CL of the light chain corresponding to the other heavy chain are amino acids that are electrically repulsive to each other, and these amino acid residues are selected from any one of the following (a) to (j): (a) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residue at position 160 in CL according to Kabat numbering; (b) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residue at position 131 in CL according to Kabat numbering; (c) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residue at position 180 in CL according to Kabat numbering; (d) the amino acid residue at position 147 in CH1 according to EU numbering and the amino acid residue at position 131 in CL according to Kabat numbering; (e) the amino acid residue at position 147 in CH1 according to EU numbering and the amino acid residue at position 160 in CL according to Kabat numbering; (f) (g) the amino acid residue at position 213 in CH1 according to EU numbering and the amino acid residue at position 123 in CL according to Kabat numbering; (h) the amino acid residues at positions 147 and 175 in CH1 according to EU numbering and the amino acid residues at positions 131 and 160 in CL according to Kabat numbering; (i) the amino acid residues at positions 147 and 175 in CH1 according to EU numbering and the amino acid residues at positions 131 and 180 in CL according to Kabat numbering; and (j) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residues at positions 131 and 160 in CL according to Kabat numbering.[B57] The cell according to any one of [B53] to [B56], wherein the electrically repulsive amino acid is (a) selected from the group consisting of lysine (K), arginine (R), and histidine (H), or (b) selected from the group consisting of glutamic acid (E) and aspartic acid (D). [B58] The cell according to any one of [B11] to [B57], wherein the antibody is a FAST-Ig (registered trademark) antibody.

[0028] [B59] The cell according to any one of [B11] to [B58], wherein the isoelectric point of one of the two different heavy chains differs from that of the other. [B60] The cell according to [B59], wherein the isoelectric point of one of the two different heavy chains differs from that of the other by 0.5 or more. [B61] The cell according to any one of [B11] to [B60], into which a mutation has been introduced that increases the difference in isoelectric point between one of the two different heavy chains and the other heavy chain. [B62] In one of the two different heavy chains, (i) amino acid residues at positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 97, 105, 108, 110, and 112 according to the Kabat numbering system in the variable region of the heavy chain, and (ii) at least one amino acid residue selected from amino acid residues at positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 (EU numbering) in the heavy chain constant region is charged; and in the other heavy chain, (i) (ii) amino acid residues at positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 97, 105, 108, 110, and 112 according to the Kabat numbering system in the heavy chain variable region; and The cell according to any one of [B11] to [B61], wherein at least one amino acid residue selected from the amino acid residues at positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 (EU numbering) in the heavy chain constant region has an opposite charge to the charged amino acid residue selected in the other heavy chain, or is uncharged.[B63] The cell according to [B62], wherein the combination of the charged amino acid residue and the amino acid residue having the opposite charge to that of the charged amino acid residue is selected from amino acid residues included in either the following group (a) or (b): (a) glutamic acid (E), aspartic acid (D); (b) lysine (K), arginine (R), histidine (H). [B64] The cell according to any of [B11] to [B63], wherein one heavy chain and the other heavy chain of the two different heavy chains are derived from IgG1 and IgG4, respectively, or IgG1 and IgG2, respectively.

[0029] [B65] The cell of any one of [B11] to [B64], wherein the antibody binds to a complex formed by HLA-DQ2.5 and a gluten peptide. [B66] The cell of [B65], wherein the antibody specifically binds to a complex formed by HLA-DQ2.5 and a gluten peptide. [B67] The cell of [B65] or [B66], wherein the antibody binds to a complex formed by HLA-DQ2.5 and a first gluten peptide and a complex formed by HLA-DQ2.5 and a second gluten peptide. [B68] The cell of any one of [B65] to [B67], wherein the antibody has substantially no binding activity to either or both of HLA-DQ2.5-positive PBMC B cells and Ba / F3 cells expressing HLA-DQ2.5. [B69] The cell according to any one of [B65] to [B68], wherein the gluten peptide is an immunodominant peptide associated with celiac disease. [B70] The cell according to any one of [B65] to [B69], wherein the gluten peptide is selected from the group consisting of a 33-mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4a gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, a hordein 1 peptide, a hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, and a 26-mer gliadin peptide, or an ω2 gliadin peptide, a BC hordein peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, a γ4a gliadin peptide, and a γ4d gliadin peptide. [B71] The cell according to any one of [B65] to [B70], wherein the antibody has substantially no binding activity to HLA-DQ2.5 in the form of a complex with a peptide unrelated to gluten peptides. [B72] The peptide unrelated to gluten peptides is a CLIP peptide, a Hepatitis B virus 1 peptide, a Salmonella peptide, a Mycobacterium bovis peptide, or a Mycobacterium[B71] The cell according to [B71], wherein the antibody is at least one peptide selected from the group consisting of HLA-DQ2.5 (HLA-DQ2.5) peptide, thyroxidase peptide, and thyroxidase peptide. [B73] The cell according to any one of [B65] to [B72], wherein the antibody is a humanized antibody. [B74] The cell according to any one of [B65] to [B73], wherein the amino acid sequence of the antibody has been modified so that the binding activity to a complex formed by HLA-DQ2.5 and a gluten peptide is enhanced. [B75] The cell according to [B74], wherein the gluten peptide contained in the complex is selected from the group consisting of ω2 gliadin peptide, BC hordein peptide, γ1 gliadin peptide, γ2 gliadin peptide, γ4a gliadin peptide, and γ4d gliadin peptide. [B76] The cell according to any one of [B65] to [B75], wherein one, two, three, or all of the sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in the following (a) to (d) in the heavy chain and light chain of the antibody have been modified to amino acid residues that are electrostatically repulsive to each other: (a) the amino acid residue in the heavy chain constant region (CH1) that is at position 175 according to EU numbering and the amino acid residue in the light chain constant region (CL) that is at position 131 according to Kabat numbering, (b) the amino acid residue in CH1 that is at position 175 according to EU numbering and the amino acid residue in CL that is at position 160 according to Kabat numbering, (c) the amino acid residue in CH1 that is at position 175 according to EU numbering and the amino acid residues in CL that are at positions 131 and 160 according to Kabat numbering, (d) The amino acid residues in CH1 are at positions 147 and 175 according to EU numbering, and the amino acid residues in CL are at positions 131 and 160 according to Kabat numbering. [B77] The cell according to any of [B65] to [B76], wherein two or more amino acid residues forming an interface between the heavy chain variable region and the light chain variable region of the antibody are amino acid residues that electrostatically repel each other.[B78] The cell according to [B77], wherein the mutually electrostatically repulsive amino acid residues are one or two sets of amino acid residues selected from the group consisting of the following sets of amino acid residues (a) and (b): (a) an amino acid residue in the heavy chain variable region that is at position 39 according to the Kabat numbering, and an amino acid residue in the light chain variable region that is at position 38 according to the Kabat numbering, (b) an amino acid residue in the heavy chain variable region that is at position 45 according to the Kabat numbering, and an amino acid residue in the light chain variable region that is at position 44 according to the Kabat numbering. [B79] The cell according to any of [B76] to [B78], wherein the mutually electrostatically repulsive amino acid residues are selected from amino acid residues included in either of the following sets (X) or (Y): (X) glutamic acid (E), aspartic acid (D), (Y) lysine (K), arginine (R), histidine (H). [B80] The cell of any of [B65] to [B79], wherein the antibody comprises an Fc domain. [B81] The cell of any of [B65] to [B80], wherein the antibody comprises an Fc domain that exhibits reduced binding affinity for human Fcγ receptors compared to a native human IgG1 Fc domain. [B82] The cell of [B80] or [B81], wherein the Fc domain comprises Arg at positions 235 and 236 (EU numbering). [B83] The cell of any of [B80] to [B82], wherein the Fc domain comprises the following (e1) or (e2): (e1) a first Fc region subunit comprising Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407 (EU numbering), and a second Fc region subunit comprising Cys at position 354 and Trp at position 366; (e2) a first Fc region subunit comprising Glu at position 439 (EU numbering), and a second Fc region subunit comprising Lys at position 356. [B84] The cell of any of [B80] to [B83], wherein the Fc domain exhibits stronger FcRn-binding affinity for human FcRn compared to native human IgG1 Fc domain.[B85] The cell of any one of [B80] to [B84], wherein the first and / or second Fc region subunit of the Fc domain comprises, according to EU numbering, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440. [B86] The cell of any one of [B80] to [B84], wherein the first and / or second Fc region subunit of the Fc domain comprises, according to EU numbering, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440. [B87] The cell according to any one of [B65] to [B86], wherein the antibody comprises one or more of the following amino acid residues (i) to (xii): (i) a glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region; (ii) a glutamic acid at position 147 (EU numbering) in the heavy chain constant region; (iii) a glutamic acid or lysine at position 131 (Kabat numbering) in the light chain constant region; (iv) a glutamic acid or lysine at position 160 (Kabat numbering) in the light chain constant region; (v) an arginine at position 235 (EU numbering) in the heavy chain constant region; (vi) an arginine at position 236 (EU numbering) in the heavy chain constant region; (vii) a lysine at position 356 (EU numbering) in the heavy chain constant region; (viii) (ix) alanine at position 434 (EU numbering) in the heavy chain constant region; (x) arginine at position 438 (EU numbering) in the heavy chain constant region; (xi) glutamic acid at position 439 (EU numbering) in the heavy chain constant region; (xii) glutamic acid at position 440 (EU numbering) in the heavy chain constant region. [B88] The cell according to any one of [B65] to [B87], wherein the antibody is a bispecific antibody comprising: a first heavy chain comprising lysine at position 175 (EU numbering), arginine at position 235 (EU numbering), arginine at position 236 (EU numbering), leucine at position 428 (EU numbering), alanine at position 434 (EU numbering), arginine at position 438 (EU numbering), glutamic acid at position 439 (EU numbering), and glutamic acid at position 440 (EU numbering);a first light chain comprising a glutamic acid at position 131 (Kabat numbering) and a glutamic acid at position 160 (Kabat numbering); a second heavy chain comprising a glutamic acid at position 147 (EU numbering), a glutamic acid at position 175 (EU numbering), an arginine at position 235 (EU numbering), an arginine at position 236 (EU numbering), a lysine at position 356 (EU numbering), a leucine at position 428 (EU numbering), an alanine at position 434 (EU numbering), an arginine at position 438 (EU numbering), and a glutamic acid at position 440 (EU numbering); and a second light chain comprising a lysine at position 131 (Kabat numbering) and a lysine at position 160 (Kabat numbering). [B89] The cell of any of [B65] to [B88], wherein the antibody's first heavy chain further comprises a glutamic acid at position 419 (EU numbering), a proline at position 445 (EU numbering), and amino acid deletions at positions 446 and 447 (EU numbering); and the second heavy chain further comprises a lysine at position 196 (EU numbering), a proline at position 445 (EU numbering), and amino acid deletions at positions 446 and 447 (EU numbering). [B90] The antibody, wherein the first heavy chain further contains a glycine at position 16 (Kabat numbering), an alanine at position 32 (Kabat numbering), a lysine at position 61 (Kabat numbering), a valine at position 35a (Kabat numbering), an alanine at position 50 (Kabat numbering), a glutamic acid at position 64 (Kabat numbering), a threonine at position 73 (Kabat numbering), a glutamic acid at position 95 (Kabat numbering), and a valine at position 102 (Kabat numbering); the first light chain further comprises a glutamic acid at position 28 (Kabat numbering), a tyrosine at position 55 (Kabat numbering), a glutamic acid or tyrosine at position 56 (Kabat numbering), a glutamic acid at position 92 (Kabat numbering), a valine at position 94 (Kabat numbering), and an alanine at position 95a (Kabat numbering);the second heavy chain has a glutamic acid at position 28 (Kabat numbering), an alanine or glutamic acid at position 30 (Kabat numbering), a glutamic acid at position 31 (Kabat numbering), a tryptophan at position 32 (Kabat numbering), a phenylalanine at position 34 (Kabat numbering), a methionine at position 35 (Kabat numbering), a serine at position 35a (Kabat numbering), a serine at position 50 (Kabat numbering), and a glutamic acid or glycine at position 61 (Kabat numbering). The cell according to any one of [B65] to [B89], further comprising a glutamic acid at position 64 (Kabat numbering), and a glutamic acid at position 65 (Kabat numbering); and the second light chain further comprising a threonine at position 25 (Kabat numbering), a lysine at position 54 (Kabat numbering), a glutamic acid at position 56 (Kabat numbering), a leucine at position 67 (Kabat numbering), a glutamine at position 79 (Kabat numbering), and a lysine at position 94 (Kabat numbering). [B91] The cell according to any one of [B65] to [B90], wherein the antibody is a bispecific antibody described in WO2022 / 059766, and is any one of the following bispecific antibodies: (1) DQN0344H0976 / L0591 / / DQN0385H1270 / L0722-F6 (a bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1270 / L0722) (2) DQN0344H0976 / L0591 / / DQN0385H1270 / L0681-F6 (a bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1270 / L0681) (3) DQN0344H0976 / L0591 / / DQN0385H1352 / L0681-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1352 / L0681) (4) DQN0344H0976 / L0591 / / DQN0385H1527 / L0605-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1527 / L0605) (5)DQN0344H0976 / L0591 / / DQN0385H1255 / L0605-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1255 / L0605) (6) DQN0344H1013 / L0620 / / DQN0385H1270 / L0722-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1270 / L0722) (7) DQN0344H1013 / L0620 / / DQN0385H1521 / L0605-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1521 / L0605) (8) DQN0344H1013 / L0620 / / DQN0385H1270 / L0681-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1270 / L0681) (9) DQN0344H1013 / L0620 / / DQN0385H1352 / L0681-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1352 / L0681) (10) DQN0344H1013 / L0620 / / DQN0385H1353 / L0681-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1353 / L0681) (11) DQN0344H0976 / L0591 / / DQN0385H1521 / L0605-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1521 / L0605) (12) DQN0344H0976 / L0591 / / DQN0385H1353 / L0681-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1353 / L0681) (13) DQN0344H1013 / L0620 / / DQN0385H1255 / L0605-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1255 / L0605) (14) DQN0344H1013 / L0620 / / DQN0385H1527 / L0605-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1527 / L0605) (15)A first polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the first heavy chain sequence of the bispecific antibody of any one of (1) to (14); a second polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the first light chain sequence of the bispecific antibody of any one of (1) to (14); any one of (1) to (14). a third polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the second heavy chain sequence of the bispecific antibody of (1); and a fourth polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the second light chain sequence of the bispecific antibody of any one of (1) to (14). [B92] The cell according to any one of [B65] to [B91], wherein the antibody is a bispecific antibody comprising a combination of four polypeptide chains selected from the group consisting of the following (1) to (15): (1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second light chain comprising the amino acid sequence of SEQ ID NO: 6; (2) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (3) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 8 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (4) (5) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10;(6) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second light chain comprising the amino acid sequence of SEQ ID NO: 6; (7) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; (8) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (9) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 8 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (10) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (11) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; (12) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (13) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; (14)a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; and (15) A first polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a first heavy chain sequence described in any one of (1) to (14); a second polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a first light chain sequence described in any one of (1) to (14); a third polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a second heavy chain sequence described in any one of (1) to (14); and a fourth polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a second light chain sequence described in any one of (1) to (14). [B93] The cell according to any one of [B65] to [B92], wherein the antibody is a bispecific antibody comprising a combination of four polypeptide chains selected from the group consisting of the following (1) to (2): (1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a first light chain comprising the amino acid sequence of SEQ ID NO: 15, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a second light chain comprising the amino acid sequence of SEQ ID NO: 17, or (2) a first polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the first heavy chain sequence described in (1); and a second polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the first light chain sequence described in (1); a third polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the second heavy chain sequence set forth in (1); anda fourth polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the second light chain sequence set forth in

[0030] [B94] The cell of any one of [B1] to [B93], comprising two or more copies of each of the four coding regions. [B95] The cell of any one of [B1] to [B93], comprising one copy of each of the four coding regions. [B96] The cell of any one of [B1] to [B93], comprising two copies of each of the four coding regions. [B97] The cell of any one of [B1] to [B93], comprising three copies of each of the four coding regions. [B98] The cell of any one of [B1] to [B93], comprising four copies of each of the four coding regions.

[0031] [B99] The cell according to any one of [B1] to [B98], wherein the vector or foreign nucleic acid has been transiently introduced. [B100] The cell according to any one of [B1] to [B99], wherein the vector or foreign nucleic acid has been episomally introduced. [B101] The cell according to any one of [B1] to [B98], wherein the vector or foreign nucleic acid has been stably introduced. [B102] The cell according to any one of [B1] to [B98] and [B101], wherein the four coding regions are contained in the genome of the cell. [B103] The cell according to [B102], wherein the four coding regions are inserted at least at the same site in the genome. [B104] The cell according to any one of [B1] to [B98] and [B101] to [B103], wherein the foreign nucleic acid has been introduced into a hotspot in the genome of the cell. [B105] The cell of any of [B1] to [B98] and [B101] to [B104], which does not contain a genomic fragment of more than 10 kb in total between the four coding regions. [B106] The cell of any of [B1] to [B98] and [B101] to [B105], which does not contain a genomic fragment of more than 5 kb, more than 4 kb, more than 3 kb, more than 2 kb, or more than 1 kb in total between the four coding regions. [B107] The cell of any of [B1] to [B98] and [B101] to [B106], which does not contain a genomic fragment of more than 5 kb, more than 4 kb, more than 3 kb, more than 2 kb, or more than 1 kb in total between the four coding regions.

[0032] [B108] The cell according to any one of [B1] to [B107], wherein the four coding regions are transcribed as different mRNA molecules. [B109] The cell according to any one of [B1] to [B108], wherein the four coding regions are transcribed as independent transcription units. [B110] The cell according to any one of [B1] to [B109], wherein the sense strands of the four coding regions are encoded on the same strand of the cell's genome. [B111] The cell according to any one of [B1] to [B110], wherein the four coding regions are transcribed from independent expression units. [B112] The cell according to [B111], wherein the expression units comprise a promoter, a coding sequence, and a poly(A) addition signal. [B113] The cell according to any one of [B1] to [B112], wherein each of the four coding regions is linked to a promoter. [B114] The cell according to [B113], wherein the promoters linked to each are the same promoter. [B115] The cell according to any one of [B112] to [B114], wherein the promoter is a pol II promoter. [B116] The cell according to any one of [B112] to [B115], wherein the promoter is selected from the group consisting of a CMV promoter, a CAG promoter, an EF1a promoter, an RSV promoter, and an SV40 promoter. [B117] The cell according to any one of [B112] to [B116], wherein the promoter is an actin promoter. [B118] The cell according to [B117], wherein the promoter is a β-actin promoter. [B119] The cell according to [B118], wherein the promoter is a chicken β-actin promoter. [B120] The cell according to any one of [B112] to [B119], wherein the promoter further comprises an enhancer. [B121] The cell according to [B120], wherein the enhancer is a CMV-IE enhancer. [B122] The cell according to any one of [B112] to [B121], wherein the promoter is a CAG promoter.

[0033] [B123] The cell according to any one of [B1] to [B122], which is a prokaryotic or eukaryotic cell. [B124] The cell according to any one of [B1] to [B123], which is a eukaryotic cell. [B125] The cell according to any one of [B1] to [B124], which is an animal cell. [B126] The cell according to any one of [B1] to [B125], which is a mammalian cell. [B127] The cell according to any one of [B1] to [B126], which is a rodent cell. [B128] The cell according to any one of [B1] to [B127], which is a Chinese hamster ovary (CHO) cell. [B129] The cell according to any one of [B1] to [B128], which is a cultured cell. [B130] The cell according to any one of [B1] to [B129], which is an in vitro cell. [B131] The cell according to any one of [B1] to [B127] and [B129], which is an ex vivo cell. [B132] The cell according to any one of [B1] to [B129], which is a cell line.

[0034] [C1] A method for producing an expression product from the vector or nucleic acid, comprising the step of expressing the vector or nucleic acid in the cell of any of [B1] to [B132]. [C2] A method for producing an expression product from the vector or nucleic acid, comprising the step of culturing the cell of any of [B1] to [B132]. [C3] A method for producing an expression product from the vector, comprising the step of introducing the vector of any of [A1] to [A105] into a cell. [C4] A method for producing an expression product from the vector or nucleic acid, comprising the steps of obtaining the cell of any of [B1] to [B132] by introducing foreign nucleic acids or vectors encoding two different heavy chains of the antibody or antigen-binding fragments thereof and two different light chains or antigen-binding fragments thereof corresponding to each heavy chain into the cell, and expressing the nucleic acids or vectors in the cell. [C5] The method of any of [C1] to [C4], further comprising the step of recovering the expression product.

[0035] [C6] A method for producing a molecule comprising an antibody or antigen-binding fragment, comprising the step of expressing the vector or nucleic acid in the cell of any of [B1] to [B132]. [C7] A method for producing a molecule comprising an antibody or antigen-binding fragment, comprising the step of culturing the cell of any of [B1] to [B132]. [C8] A method for producing a molecule comprising an antibody or antigen-binding fragment, comprising the step of introducing the vector of any of [A1] to [A105] into a cell. [C9] A method for producing a molecule comprising an antibody or antigen-binding fragment, comprising the steps of obtaining the cell of any of [B1] to [B132] by introducing foreign nucleic acids or vectors encoding two different heavy chains of the antibody or antigen-binding fragments thereof and two different light chains or antigen-binding fragments thereof corresponding to each heavy chain into the cell, and expressing the nucleic acids or vectors in the cell. [C10] The method of any of [C6] to [C9], further comprising the step of recovering the molecule comprising the produced antibody or antigen-binding fragment.

[0036] [D1] An expression product produced by the method of any one of [C1] to [C5]. [D2] A composition comprising an expression product produced by the method of any one of [C1] to [C5]. [D3] A molecule comprising an antibody or an antigen-binding fragment produced by the method of any one of [C6] to [C10]. [D4] A composition comprising a molecule comprising an antibody or an antigen-binding fragment produced by the method of any one of [C6] to [C10].

[0037] The present invention also includes the following: [1] A recombinant vector comprising four coding regions in a 1:1:1:1:1 ratio, each coding region encoding two different antibody heavy chains or antigen-binding fragments thereof and two different light chains or antigen-binding fragments thereof corresponding to each heavy chain. [2] The vector according to [1], wherein the antibody is a multispecific antibody. [3] The vector according to [1] or [2], wherein pairing between one heavy chain of the antibody and pairing between the other heavy chains are inhibited by steric hindrance due to knob-into-hole and / or charge repulsion of charged amino acids. [4] The vector according to any of [1] to [3], wherein pairing between one heavy chain of the antibody and the light chain corresponding to the other heavy chain, and pairing between the other heavy chain and the light chain corresponding to the one heavy chain are inhibited by charge repulsion of charged amino acids. [5] The vector according to any one of [1] to [4], wherein the antibody binds to a complex formed by HLA-DQ2.5 and a gluten peptide. [6] The vector according to any one of [1] to [5], comprising two copies of each of the four coding regions. [7] The vector according to any one of [1] to [6], wherein the four coding regions are transcribed as different mRNA molecules. [8] The vector according to [7], wherein a promoter is linked to each of the four coding regions. [9] The vector according to any one of [1] to [8], wherein the vector is a mammalian expression vector.

[10] A cell into which the vector according to any one of [1] to [9] has been introduced.

[11] The cell according to

[10] , wherein the vector has been introduced into the genome of the cell.

[12] The cell according to

[10] , wherein two copies of the vector have been introduced into the genome.

[13] The cell according to any one of

[10] to

[12] , wherein the genome contains four copies of each of the four coding regions.

[14] The cell of any one of

[10] to

[13] , which is a mammalian cell.

[15] A method for producing a molecule containing an antibody or an antigen-binding fragment, comprising the step of expressing the vector in the cell of any one of

[10] to

[14] .

[16] The method of

[15] , further comprising the step of recovering the expression product.

[0038] The present invention enables efficient production of recombinant antibodies. The present invention is useful for producing desired recombinant antibodies and polypeptides containing antigen-binding fragments thereof. It is particularly useful for producing molecules containing multispecific antibodies and antigen-binding fragments thereof.

[0039] Figure 1 shows the results of measuring antibody production and the proportion of bispecific antibodies in the produced antibodies after transfection of cells (n = 3) with expression vectors encoding three bispecific antibodies (3CC1, 3CC2, and 3CC3) that bind to the HLA-DQ2.5 / gluten peptide complex at a copy number ratio of H1:L1:H2:L2 = 2:2:2:2 or 1:2:2:3. The H1:L1:H2:L2 = 2:2:2:2 expression vector contains two copies of each of four expression units, each flanked by a CAG promoter and poly(A) signal, coding for the first heavy chain (H1) and its associated light chain (L1), the second heavy chain (H2) and its associated light chain (L2). The H1:L1:H2:L2 = 1:2:2:3 expression vector contains one, two, two, or three copies, respectively. The plasmid also contains the DHFR gene as a selectable marker and an origin of replication (ori) for amplification in E. coli. Results are presented as box-and-whisker plots. The boxes represent the interquartile range, with the lower limit of the box representing the first quartile (25th percentile) and the upper limit representing the third quartile (75th percentile). The open bars represent the median. The upper and lower whiskers represent the maximum and minimum values, respectively.

[0040] Figure 2 compares antibody production from expression vectors encoding three bispecific antibodies (3CC1, 3CC2, and 3CC3) that bind to the HLA-DQ2.5 / gluten peptide complex at the copy number ratios shown in Figure 1, as well as at copy number ratios of H1:L1:H2:L2 = 1:1:1:1 and 1:3:1:3. Each expression vector was introduced into cells (n = 3), and the amount of antibody produced and the percentage of bispecific antibodies among the produced antibodies were measured. The results are shown in box plots.

[0041] The present invention provides a method for producing a polypeptide comprising an antibody or its antigen-binding fragment using cells containing four coding regions encoding two types of antibody heavy chains or antigen-binding fragments thereof and two types of light chains or antigen-binding fragments thereof corresponding to each heavy chain, in a copy number ratio of 1:1:1:1, as well as vectors and cells useful for this method. Hereinafter, embodiments of the present invention will be described in more detail. Hereinafter, a polypeptide comprising an antibody or its antigen-binding fragment may be referred to as an antigen-binding molecule. Herein, the two types of heavy chains or antigen-binding fragments thereof, and the two types of light chains or antigen-binding fragments thereof are separate polypeptide chains, and the four coding regions encoding them are independent coding regions (i.e., separated coding regions).

[0042] First, as used herein, "and / or" refers to the listed elements alone and in any combination. Specifically, for example, "A and / or B" refers to the listed elements A and B alone and in any combination, specifically (1) A, (2) B, and (3) A and B. "A, B, and / or C" refers to the listed elements A, B, and C alone and in any combination, including the following variations: (1) A, (2) B, (3) C, (4) A and B, (5) A and C, (6) B and C, and (7) A, B, and C.

[0043] As used herein, the terms "first," "second," "third," etc. are used for the convenience of distinguishing between two or more different types of moieties. The use of these terms is not intended to confer a specific order or orientation unless otherwise specified. For example, with respect to antibody heavy chains, light chains, antigen-binding fragments thereof, etc., the terms "first," "second," "third," etc. are used herein for the convenience of distinguishing between two or more different types of moieties, and are not intended to confer a specific order or orientation unless otherwise specified. The order may be arbitrarily reversed as appropriate. For example, what is referred to as "first" may be referred to as "second," and what is referred to as "second" may be referred to as "first."

[0044] For example, an antibody having two types of heavy chains and two types of light chains has a first heavy chain and a first light chain paired with it, and a second heavy chain and a second light chain paired with it. In this case, the second heavy chain and light chain are referred to as the first heavy chain and light chain, and the first heavy chain and light chain are referred to as the second heavy chain and light chain.

[0045] In the present invention, "two types," "four types," etc. are used to mean "two different" and "four different," respectively. That is, "two types of heavy chains" refer to "two different heavy chains," and each heavy chain has a different structure (amino acid sequence). "Two types of light chains" refer to "two different light chains," and each light chain has a different structure (amino acid sequence). In the present invention, the first heavy chain and the second heavy chain are different from each other, i.e., they have different structures, specifically, different amino acid sequences. Furthermore, the first light chain and the second light chain are also different from each other, i.e., they have different structures, specifically, different amino acid sequences. The "four coding sequences" encoding the "two types of heavy chains" and "two types of light chains" are different from each other.

[0046] Unless otherwise indicated, amino acid residues in the light chain constant region are numbered herein according to Kabat et al., and numbering of amino acid residues in the heavy chain constant region is according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The EU numbering system is also referred to as the EU index.

[0047] As used herein, amino acids are described by one-letter or three-letter codes or both, for example, Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, or Val / V.

[0048] Antibodies and molecules containing fragments thereof produced in the present invention may contain amino acid modifications (also referred to herein as "amino acid substitutions" or "amino acid mutations") to the amino acid sequence of a natural antibody. For amino acid modifications, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed. Furthermore, several known methods for amino acid modification, such as substituting non-natural amino acids, can also be employed (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA carrying an unnatural amino acid bound to a complementary amber suppressor tRNA for the UAG codon (amber codon), a type of stop codon, is suitable. Modifying an amino acid is also called introducing an amino acid mutation.

[0049] Furthermore, in the present specification, expressions indicating amino acid modifications may be appropriately used in which the one-letter or three-letter code of the amino acid before and after the modification is indicated before and after the number indicating a specific position. For example, the modification T366W or Thr366Trp (EU numbering) used to substitute an amino acid contained in the CH3 region of an antibody indicates a substitution of Thr at position 366 (EU numbering) with Trp. That is, the number indicates the amino acid position according to EU numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution. Furthermore, those represented by Kabat numbering indicate substitutions in Kabat numbering.

[0050] Furthermore, the antibodies produced in the present invention are not limited to natural antibodies but may also be artificially produced molecules. In the present invention, antibodies are not limited to monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific or trispecific antibodies), antibody fragments, crossover Fabs or cross Fabs, and cross Mabs, as long as they exhibit the desired antigen-binding activity. In the present invention, antibodies preferably contain four types of chains: two heavy chains or fragments thereof, and two light chains or fragments thereof. Linker sequences, tag sequences, cleavage sequences, etc. may be added to the antibodies as appropriate.

[0051] For example, a polypeptide tagged with glutathione S-transferase (GST) can be purified using a glutathione column. Tagging is not limited to GST tagging, and may be performed with any tag, including, but not limited to, histidine tag, MBP, CBP, Flag tag, HA tag, V5 tag, c-myc tag, etc. The antibody of the present invention may contain such a tag.

[0052] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies (e.g., variant antibodies containing naturally occurring mutations or variant antibodies that arise during the production of a monoclonal antibody preparation, which are typically present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies are typically produced by a variety of techniques, including hybridoma technology, recombinant DNA technology, phage display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods for making monoclonal antibodies are well known to those of skill in the art.

[0053] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain including an Fc region. A "full length antibody," "complete antibody," and "whole antibody" typically comprises two heavy chains each comprising a heavy chain variable region (VH) and CH1, CH2, and CH3 domains of the heavy chain constant region, and two light chains each comprising a light chain variable region (VL) and a CL domain of the light chain constant region. The antibodies produced in the present invention may be "full length antibodies," "complete antibodies," or "whole antibodies."

[0054] The antibodies of the present invention are preferably "tetrameric antibodies." A "tetrameric antibody" is an antibody that comprises a tetramer containing two heavy chains and two light chains derived from a "natural antibody." The term "natural antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain contains a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of its constant domain. However, the tetrameric antibody is not limited to a natural antibody, but may be a modified antibody or an artificially produced antibody.

[0055] The antibodies produced in the present invention may also be immunoglobulin molecules. "Immunoglobulin molecule" refers to a protein having the structure of an antibody. For example, IgG class immunoglobulins are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has, from N- to C-terminus, a variable region (VH), also known as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also known as a heavy chain constant region. Similarly, each light chain has, from N- to C-terminus, a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a constant light (CL) domain, also known as a light chain constant region. Immunoglobulin heavy chains may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further classified into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains may be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and an Fc domain linked via an immunoglobulin hinge region.

[0056] The term "hinge region" refers to the region where two antibody heavy chains form disulfide bonds. It is known that disulfide bonds are formed between the cysteine ​​residues at positions 226 and 229 in the hinge region (EU numbering) of two antibody heavy chains. The hinge region may be the region from positions 226 to 229 (EU numbering) or positions 239 to 242 (Kabat numbering). Alternatively, it may be the region called the middle hinge, from positions 226 to 230 (EU numbering) or positions 239 to 243 (Kabat numbering). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by arranging the first and last cysteine ​​residues that form inter-heavy chain disulfide bonds in the same positions (see, for example, Table 1 in Brekke et al., 1995, Immunol Today 16: 85-90). Hinge regions as used herein include wild-type hinge regions, as well as variants in which amino acid residues in the wild-type hinge region have been altered by substitution, addition, or deletion.

[0057] In one embodiment, the antibody of the present invention may have an amino acid residue in its hinge region substituted with at least one cysteine ​​residue, which may be at EU numbering positions 226 and / or 229 in the hinge region.

[0058] In the present invention, the antibody may also have a disulfide bond formed between amino acids not located in the hinge region. In some embodiments, such a disulfide bond is formed, connected, or linked through an amino acid located at a position selected from among positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 (EU numbering) in the CH1 region. In a preferred embodiment, such a disulfide bond is formed, connected, or linked through an amino acid located at position 191 (EU numbering) in the CH1 region. For example, the CH1 region contains a cysteine ​​residue at position 191 (EU numbering) (via mutation, substitution, or insertion).

[0059] In the present invention, antibodies may be of any class. The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes). For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Particularly useful light chain constant regions include either of two isotypes: κ and λ.

[0060] In one embodiment, the constant region of the antibody of the present invention is of human origin. In a specific embodiment, the subclass of the heavy chain constant region is any of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In a specific embodiment, the subclass of the CH1 region is any of γ1, γ2, γ3, γ4, α1, α2, μ, δ, and ε.

[0061] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0062] The term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR"), and / or forms structurally defined loops (the "hypervariable loops"), and / or contains antigen-contacting residues (the "antigen contact groups"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3).

[0063] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0064] The antibodies produced in the present invention may or may not contain a constant region. The constant region is preferably an antibody constant region, more preferably an IgG1, IgG2, IgG3, or IgG4 antibody constant region, and even more preferably an antibody constant region of human IgG1, IgG2, IgG3, or IgG4. The constant region is preferably a heavy chain constant region, more preferably an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, and even more preferably an antibody constant region of human IgG1, IgG2, IgG3, or IgG4. The amino acid sequences of the human IgG1 constant region, human IgG2 constant region, human IgG3 constant region, and human IgG4 constant region are known. For the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4, multiple allotype sequences due to genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, and any of these can be used in the present invention. The constant region with altered amino acids may contain other amino acid mutations or modifications, as long as it contains the amino acid mutation of the present invention.

[0065] The antibodies of the present invention may or may not contain an Fc region. In a preferred embodiment, the antibodies of the present invention contain an Fc region. In the present invention, the term "Fc region" or "Fc domain" is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. An "Fc region" or "Fc domain" refers to the region of an antibody molecule comprising a hinge or a portion thereof, and a fragment consisting of the CH2 and CH3 domains. The Fc region of an IgG class refers, for example, but is not limited to, the region from cysteine ​​226 (EU numbering, also referred to herein as the EU index) to the C-terminus, or from proline 230 (EU numbering) to the C-terminus. The C-terminal lysine (Lys447) or glycine-lysine (residues Gly446-Lys447) of the Fc region may or may not be present. The Fc region can be preferably obtained, for example, by partially digesting an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody with a protease such as pepsin, followed by re-elution of the fraction adsorbed to a Protein A column or Protein G column. Such a protease is not particularly limited, as long as it can digest a full-length antibody to form Fab or F(ab')2 exclusively under appropriately selected enzyme reaction conditions (e.g., pH). Examples include pepsin and papain.

[0066] For example, an Fc region derived from a native IgG can be used as the "Fc region." Here, native IgG refers to a polypeptide that contains the same amino acid sequence as an IgG found in nature and belongs to the class of antibodies substantially encoded by the immunoglobulin gamma gene. Native human IgG refers to, for example, native human IgG1, native human IgG2, native human IgG3, or native human IgG4. Native IgG also includes naturally occurring variants thereof. Multiple allotype sequences based on genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, for the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, and any of these sequences can be used in the present invention. In particular, the sequence of human IgG1 may have Asp-Glu-Leu (DEL) or Glu-Glu-Met (EEM) as the amino acid sequence at positions 356 to 358 (EU numbering).

[0067] In some embodiments, the Fc domain of the antibody of the present invention is composed of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other. Although the antibody of the present invention may not comprise an Fc domain, in one embodiment, the antibody of the present invention comprises one Fc domain. Each subunit of the Fc domain is usually linked to the C-terminus of each heavy chain variable region.

[0068] As used herein, the Fc domain of the antibody of the present invention is preferably an IgG Fc domain. In some preferred embodiments, the Fc domain is a human IgG Fc domain. In another preferred embodiment, the Fc domain is an IgG1 Fc domain. In a further preferred embodiment, the Fc domain is a human IgG1 Fc domain.

[0069] The antibodies of the present invention may be chimeric antibodies. The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. Similarly, the term "chimeric antibody variable domain" refers to an antibody variable region in which a portion of the heavy and / or light chain variable region is derived from a particular source or species, while the remainder of the heavy and / or light chain variable region is derived from a different source or species.

[0070] The antibody of the present invention may be a humanized antibody. A "humanized" antibody refers to a chimeric antibody containing amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. A "humanized antibody variable region" refers to the variable region of a humanized antibody.

[0071] Humanized antibodies and methods for their production are also reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991). (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guide selection" approach for FR shuffling).

[0072] The antibody of the present invention may be a human antibody. A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that uses a human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. A "human antibody variable region" refers to the variable region of a human antibody.

[0073] Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0074] The present invention also encompasses the production of antibody fragments. An "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. An antibody fragment may also be considered an antibody. An example of such an antibody fragment includes F(ab')2. For reviews of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003); Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); and, in addition, WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives, see U.S. Patent No. 5,869,046. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies as described herein, or production of recombinant antibodies in host cells (e.g., prokaryotic cells such as E. coli, or eukaryotic cells such as animal cells).

[0075] As used herein, the terms "antigen-binding fragment," "antigen-binding portion," and "antigen-binding domain" of an antibody refer to a portion of the antibody that specifically binds to a portion or all of an antigen. When an antigen has a large molecular weight, the antigen-binding fragment, antigen-binding portion, and antigen-binding domain may bind only to a specific portion of the antigen. This specific portion is called an epitope. The antigen-binding domain of an antibody includes an antibody fragment that binds to a specific antigen. The antigen-binding fragment, antigen-binding portion, and antigen-binding domain may be composed of, for example, one or more antibody variable regions. The antigen-binding fragment of an antibody may comprise four chains. Specifically, it may comprise two antibody heavy chains or fragments thereof and two antibody light chains or fragments thereof, for a total of four chains. In a non-limiting embodiment, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding domains include, but are not limited to, F(ab')2. In one embodiment, the antigen-binding domain comprises an Fab comprising a CH1 region. For example, it may comprise two Fabs. In some embodiments, the antigen-binding domain comprises an antibody hinge region. Alternatively, the antigen-binding domain may not comprise a hinge region, as long as two heavy chain fragments containing the CH1 region form a dimer. For example, a polypeptide containing a heavy chain fragment containing the CH1 region and a dimerization sequence can be used.

[0076] "F(ab')2" consists of two light chains and two heavy chains containing partial constant regions, such as the CH1 and CH2 domains, such that disulfide bonds are formed between the two heavy chains. The F(ab')2 disclosed herein can be conveniently prepared by partially digesting a full-length monoclonal antibody or the like having the desired antigen-binding domain with a protease such as pepsin, followed by removal of the Fc fragment by adsorption onto a protein A column. The protease is not particularly limited, as long as it can cleave the full-length antibody to produce F(ab')2 in a limited manner by appropriately setting the enzymatic reaction conditions, such as pH. Examples of such proteases include pepsin and ficin. However, the F(ab')2 of the present invention is not limited to molecules produced in this manner; it may also be an artificially produced molecule, such as a recombinant molecule, as long as it has an equivalent structure. Furthermore, the F(ab')2 may be a multispecific (bispecific) molecule.

[0077] The terms "multispecific" and "bispecific" refer to an antigen-binding molecule, such as an antibody, that can specifically bind to at least two different antigenic determinants. The antigenic determinants may be on the same antigen molecule or on different antigenic molecules. For example, a bispecific antibody of the present invention may contain a moiety capable of binding to a different antigenic determinant, as long as it is capable of binding to two different antigenic determinants via two heavy chains and two light chains. Such an additional antigen-binding moiety may be, for example, a single-chain Fv (scFv), a single-chain antibody, a single-chain Fv2 (scFv2), a single-domain antibody, or the like, added to the antibody. For example, an additional antigen-binding moiety can be fused to the N-terminus and / or C-terminus of either of the two heavy chains and two light chains. In other words, molecules known as triabodies and tetrabodies are also included in the bispecific antibody of the present invention, as long as they contain a structure capable of binding to two different antigenic determinants via two heavy chains and two light chains. The bispecific antibody of the present invention is preferably composed of four chains, i.e., two heavy chains and two light chains.

[0078] As used herein, the term "specifically binds" refers to binding to a certain target more strongly than to other targets. For example, it may mean that no significant binding is exhibited to molecules other than the specifically bound molecule. This expression is also used when an antigen-binding domain is specific for a specific epitope among multiple epitopes contained in an antigen. When the epitopes to which the antigen-binding domain binds are contained in multiple different antigens, an antigen-binding molecule containing the antigen-binding domain can bind to various antigens containing the epitope. In other words, the target to which it specifically binds may be one or multiple. For example, if a molecule binds to A and B but does not significantly bind to anything else, the molecule is specific for A and B. In this case, the molecule may bind to A and B simultaneously, or exclusively, i.e., when it binds to A, it cannot bind to B, and when it binds to B, it cannot bind to A.

[0079] The antibody of the present invention may be a crossover Fab. A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which either the variable or constant regions of the Fab heavy chain and Fab light chain have been exchanged; i.e., the crossover Fab molecule comprises a peptide chain composed of a light chain variable region and a heavy chain constant region, and a peptide chain composed of a heavy chain variable region and a light chain constant region. For clarity, in a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the crossover Fab molecule. Conversely, in a crossover Fab molecule in which the constant regions of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0080] In contrast, a "conventional" or "native" Fab molecule refers to a Fab molecule in its native format, i.e., a Fab molecule comprising a heavy chain (VH-CH1) made up of the variable and constant regions of the heavy chain, and a light chain (VL-CL) made up of the variable and constant regions of the light chain.

[0081] When an antibody comprises an Fc region, the Fc region may contain appropriate mutations. Examples of such mutations include, but are not limited to, a mutation resulting in a deletion of amino acids 231A to 238S (EU numbering) (WO 2009 / 011941), as well as C226S, C229S, P238S, and (C220S) (J. Rheumatol (2007) 34, 11); C226S and C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54); and C226S, C229S, E233P, L234V, and L235A (Blood (2007) 109, 1185-1192).

[0082] For the Fc region of a natural IgG1, IgG2, IgG3, or IgG4 antibody, see, for example, the sequence of RefSeq accession number AAC82527.1 with A added to the N-terminus, the sequence of RefSeq accession number AAB59393.1 with A added to the N-terminus, the sequence of RefSeq accession number CAA27268.1 with A added to the N-terminus, and the sequence of RefSeq accession number AAB59394.1 with A added to the N-terminus (all of these are referred to as sequences registered in RefSeq (NCBI Reference Sequence Database) as of the earliest priority date of the present application, and can be identified by referring to the revision history of the database).

[0083] For example, these include Fc domains having at least one amino acid mutation (e.g., substitution) selected from the following amino acid positions: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, 332, 439, 445, 449, or 451 (EU numbering). The antibody isotype from which the Fc domain is derived is not particularly limited, and an appropriate Fc domain derived from a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be used. It is preferable to use an Fc domain derived from an IgG1 antibody.

[0084] In the present invention, there is no particular limitation on the antigen, and the present invention can be applied to an antibody or antigen-binding fragment thereof against a desired antigen.

[0085] In one embodiment, the antigenic molecule is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ionotropic receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD4 antigens, costimulatory molecules, MHC molecules, growth factors or their receptors, growth factors or their receptors, and cell adhesion molecules, including, but not limited to, tight junction membrane proteins, T cell receptor (TCR) complex proteins, TNFR (tumor necrosis factor receptor) superfamily members, MHC class II (HLA class II) molecules (including HLA-DR, DQ, DP, etc.), and platelet-derived growth factors (PDGFs) (including PDGF-A, PDGF-B, PDGF-C, PDGF-D, etc.) or their receptors.

[0086] Examples of cell adhesion molecules include, but are not limited to, claudin family members such as Claudin 6; examples of TCR complex-forming proteins include CD3, particularly CD3ε; examples of MHC class II (HLA class II) molecules include HLA-DQ2.5, HLA-DQ7.5, HLA-DQ5.1, HLA-DQ6.3, HLA-DQ7.3, and HLA-DQ8, particularly HLA-DQ2.5; and examples of PDGFs include PDGF-B and PDGF-D.

[0087] As mentioned above, in one embodiment, the antibody of the present invention may further comprise a third antigen-binding domain, which may be fused to either the first or second antigen-binding domain.

[0088] In one embodiment, the third antigen-binding domain can be a Fab or scFv. In this case, the third antigen-binding domain may be fused at its C-terminus to the N-terminus of the Fab heavy chain (VH region) of either the first or second antigen-binding domain, optionally via a peptide linker. For example, when the third antigen-binding domain is a Fab, the VH constituting the Fab may be fused to the N-terminus of the heavy or light chain of the first or second antigen-binding domain, and the VL constituting the Fab may be fused to the N-terminus of the light or heavy chain of the first or second antigen-binding domain. When the third antigen-binding domain is an scFv, the scFv may be fused to the N-terminus of either the heavy or light chain of the first or second antigen-binding domain.

[0089] In another embodiment, the antibody of the present invention comprises first, second, and third antigen-binding domains, each of which can be a Fab molecule. In this case, the third antigen-binding domain may be fused at the C-terminus of the Fab heavy chain (CH1 region) to the N-terminus of the Fab heavy chain (VH region) of either the first or second antigen-binding domain, optionally via a peptide linker.

[0090] In another embodiment, the antibody of the present invention comprises a third antigen-binding domain, which is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and each of the first antigen-binding domain and the second antigen-binding domain can be a conventional Fab molecule.

[0091] In another embodiment, the antibody of the present invention comprises a third antigen-binding domain capable of binding to a third antigen different from the first and second antigens described above. The third antigen-binding domain that binds to the third antigen may be an antigen-binding domain that recognizes any antigen.

[0092] In another preferred embodiment, the antibody of the present invention comprises two antigen-binding domains, each of which is a Fab, with the first antigen-binding domain fused at the C-terminus of the Fab heavy chain to the N-terminus of either the first or second Fc subunit, and the second antigen-binding domain fused at the C-terminus of the Fab heavy chain to the N-terminus of the other Fc subunit, resulting in a molecule with a structure similar to that of a native antibody.

[0093] The antibody constant region, particularly the heavy chain constant region, may be modified as necessary to improve the function or stability of the antibody. Examples of modifications to improve antibody function include modifications that strengthen or weaken the binding between the antibody and Fcγ receptor ("FcγR"), modifications that strengthen or weaken the binding between the antibody and FcRn, and modifications that strengthen or weaken the cytotoxic activity of the antibody (e.g., ADCC activity and CDC activity). In addition, modifications that improve antibody heterogeneity, and modifications that improve non-immunogenicity and / or pharmacokinetics may also be included.

[0094] Furthermore, heterogeneity in the C-terminal sequences of IgG antibodies heavy chains has been reported to be due to amidation of the C-terminal carboxyl group caused by deletion of the C-terminal amino acid lysine residue or deletion of two C-terminal amino acids, glycine and lysine (Anal. Biochem. 2007 Jan 1:360(1):75-83). Therefore, in the present invention, in order to reduce heterogeneity in the C-terminus of the heavy chain, it is preferable to use IgG in which the C-terminal lysine or the C-terminal lysine and glycine are deleted. Chimeric antibodies and humanized antibodies using human-derived sequences have reduced antigenicity in the human body and are therefore expected to be useful when administered to humans for therapeutic purposes, etc.

[0095] In some preferred embodiments, the Fc region of an antibody of the present invention is composed of a first and a second Fc region subunit capable of stable association, and in some embodiments, the Fc region exhibits reduced binding affinity to an Fcγ receptor, e.g., a human Fcγ receptor, compared to a native Fc region, e.g., a human IgG1 Fc region.

[0096] In a particular embodiment, the Fc region is composed of first and second Fc region subunits capable of stable association and exhibits reduced binding affinity for human Fcγ receptors compared to native human IgG1 Fc region, wherein the first Fc region subunit is selected from the group consisting of: (a1) an Fc region polypeptide comprising mutations L234A, L235A; (a2) an Fc region polypeptide comprising mutations L234A, L235A, N297A; and (a3) ​​an Fc region polypeptide comprising mutations L234A, L235A, N297A, S354C, T366W, and the second Fc region subunit is selected from the group consisting of: (a4) an Fc region polypeptide comprising mutations L234A, L235A; (a5) an Fc region polypeptide comprising mutations L234A, L235A, N297A; and (a6) Fc region polypeptides comprising the mutations L234A, L235A, N297A, Y349C, T366S, L368A, Y407V (amino acid positions are numbered using EU index numbering).

[0097] The antibodies to be produced in the present invention contain two different heavy chains and two different light chains. This characteristic is seen, for example, in bispecific antibodies. The present invention is preferably applied to the production of such antibodies in which the association between heavy chains is regulated and / or the association between heavy chains and light chains is regulated.

[0098] An antibody in which the two heavy chains contained therein are different is called a heterogeneous heavy chain antibody. An antibody in which the two light chains contained therein are different is called a heterogeneous light chain antibody. The antibodies to be produced in the present invention have heterogeneous heavy chains and also heterogeneous light chains. An antibody in which the two heavy chains contained therein are the same is called a homogeneous heavy chain antibody. An antibody in which the two light chains contained therein are the same is called a homogeneous light chain antibody. Natural antibodies have homogeneous heavy chains and homogeneous light chains.

[0099] For example, the present invention is preferably applied to antibodies that contain two different heavy chains and have been modified to more easily form heterodimers between different heavy chains, i.e., dimers between one heavy chain and the other heavy chain, than homodimers between the same heavy chains, i.e., homodimers of one heavy chain and the other heavy chain.

[0100] For example, in a preferred embodiment, the Fc region of an antibody of the present invention comprises a modification that promotes association of the first and second subunits of the Fc region (i.e., the subunits of the Fc region comprised in the first heavy chain and the subunits of the Fc region comprised in the second heavy chain) (the modification may also be a modification that suppresses association between the subunits of the Fc region comprised in the first heavy chain and / or a modification that suppresses association between the subunits of the Fc region comprised in the second heavy chain). In a specific embodiment, the modification is a so-called "knob-into-hole" modification that comprises a "knob" modification in one of the two subunits of the Fc region and a "hole" modification in the other of the two subunits of the Fc region, as described in more detail below.

[0101] Knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). This technology generally involves introducing a protrusion ("knob") into the interface of a first polypeptide and a corresponding cavity into the interface of a second polypeptide, so that the protrusion ("knob") can be positioned in the cavity ("hole"), promoting heterodimer formation and preventing homodimer formation. The protrusion is constructed by replacing small amino acid side chains in the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine).

[0102] Thus, in a particular embodiment, in the CH3 domain of a first subunit of an antibody Fc domain, amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be positioned in a cavity in the CH3 domain of a second subunit, and in the CH3 domain of a second subunit of the Fc domain, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can be positioned.

[0103] The protrusions and cavities can be made by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis, or by peptide synthesis.

[0104] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0105] In yet a further embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is further replaced with a cysteine ​​residue (S354C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is further replaced with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues results in the formation of disulfide bridges between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0106] For example, in the case of human IgG, an antibody in which the Y349C and T366W modifications have been made to make the amino acid side chains in the CH3 region of one H chain larger (knob), and the D356C (or E356C), T366S, L368A, and Y407V modifications have been made to make the amino acid side chains in the CH3 region of the other H chain smaller, is preferred.

[0107] In some embodiments, the Fc domain in the multispecific antibody is composed of a first Fc region subunit and a second Fc region subunit capable of stable association. In some embodiments, the Fc domain in the multispecific antibody comprises the following (e1) or (e2): (e1) a first Fc region subunit comprising Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407, and a second Fc region subunit comprising Cys at position 354 and Trp at position 366; (e2) a first Fc region subunit comprising Glu at position 439, and a second Fc region subunit comprising Lys at position 356 (amino acid positions are numbered according to EU numbering).

[0108] Furthermore, other techniques for promoting the association of desired combinations of H chains and L chains can be applied to the antibodies of the present invention.

[0109] For example, techniques for suppressing undesired association of antibody chains can be applied, such as introducing electrostatic repulsion at the interface of the second or third constant region (CH2 or CH3) of the antibody heavy chain, or modifying amino acid residues that form the hydrophobic core at the interface between the heavy chain variable region and the light chain variable region to convert them to charged polar amino acids, so as to suppress unintended association between the heavy chain and the light chain (WO2006 / 106905).

[0110] In the technique for suppressing unintended H chain association by introducing electrostatic repulsion at the CH2 or CH3 interface, examples of amino acid residues that contact the interface of the other H chain constant region include regions corresponding to residues at EU numbering positions 356, 439, 357, 370, 399, and 409 in the CH3 region.

[0111] More specifically, examples include antibodies comprising two types of H chain CH3 regions, in which one to three pairs of amino acid residues selected from the pairs of amino acid residues shown in (1) to (3) below in the first H chain CH3 region have the same electric charge: (1) the amino acid residues at EU numbering positions 356 and 439 in the H chain CH3 region, (2) the amino acid residues at EU numbering positions 357 and 370 in the H chain CH3 region, and (3) the amino acid residues at EU numbering positions 399 and 409 in the H chain CH3 region.

[0112] For example, such an H chain may be an H chain in which the amino acid residues at EU numbering positions 356 and 439 have the same charge, an H chain in which the amino acid residues at EU numbering positions 357 and 370 have the same charge, or an H chain in which the amino acid residues at EU numbering positions 399 and 409 have the same charge.

[0113] Furthermore, the antibody may be an antibody having pairs of amino acid residues in a second H chain CH3 region that are different from the first H chain CH3 region, and the pairs of amino acid residues may be selected from the pairs of amino acid residues (1) to (3) above, and one to three pairs of amino acid residues corresponding to the pairs of amino acid residues (1) to (3) that have the same electric charge in the first H chain CH3 region may have an opposite electric charge to the corresponding amino acid residues in the first H chain CH3 region.

[0114] For example, if the first H chain contains positively charged amino acids at EU numbering positions 356 and 439, the second H chain preferably contains negatively charged amino acids at EU numbering positions 356 and 439. Conversely, if the first H chain contains negatively charged amino acids at EU numbering positions 356 and 439, the second H chain preferably contains positively charged amino acids at EU numbering positions 356 and 439. If the first H chain contains positively charged amino acids at EU numbering positions 357 and 370, the second H chain preferably contains negatively charged amino acids at EU numbering positions 357 and 370. Conversely, if the first H chain contains negatively charged amino acids at EU numbering positions 357 and 370, the second H chain preferably contains positively charged amino acids at EU numbering positions 357 and 370. If the first H chain contains positively charged amino acids at EU numbering positions 399 and 409, the second H chain preferably contains negatively charged amino acids at EU numbering positions 399 and 409. Conversely, if the first H chain contains negatively charged amino acids at EU numbering positions 399 and 409, the second H chain preferably contains positively charged amino acids at EU numbering positions 399 and 409.

[0115] The amino acid residues shown in (1) to (3) above are close to each other when associated. Those skilled in the art can find positions corresponding to the amino acid residues in (1) to (3) above in the desired H chain CH3 region or H chain constant region by homology modeling using commercially available software, and can appropriately modify the amino acid residues at these positions.

[0116] In the above-mentioned antibody, the "charged amino acid residue" is preferably selected from, for example, any one of the following amino acid residues: (a) glutamic acid (E) and aspartic acid (D), and (b) lysine (K), arginine (R), and histidine (H).

[0117] In the above antibodies, the terms "amino acids with the same charge," "amino acids with the same charge," and "amino acids with electrically repulsive charges" mean, for example, that two or more amino acid residues are all amino acids selected from the amino acid residues included in either one of the above groups (a) and (b). The term "amino acids with opposite charges" means, for example, that when a certain amino acid residue is selected from the amino acid residues included in either one of the above groups (a) and (b), the amino acid is selected from the amino acid residues included in the other group.

[0118] Particularly preferred examples include antibodies in which the heavy chain of the first antibody has positively charged amino acids (e.g., lysine (K), arginine (R), or histidine (H)) at EU numbering positions 356 and 439, and the heavy chain of the second antibody has negatively charged amino acids (e.g., glutamic acid (E) or aspartic acid (D)) at EU numbering positions 356 and 439. When the heavy chain of a native antibody has a negatively charged amino acid at EU numbering 356 (e.g., glutamic acid (E) or aspartic acid (D)) and a positively charged amino acid at EU numbering 439 (e.g., lysine (K), arginine (R), or histidine (H)), it is preferable to substitute, for example, the amino acid at EU numbering 356 in the heavy chain of a first antibody with a positively charged amino acid (e.g., lysine (K), arginine (R), or histidine (H)) and the amino acid at EU numbering 439 in the heavy chain of a second antibody with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)). As described above, the first antibody and the second antibody are interchangeable, and the above description also encompasses substitution of amino acid at EU numbering 439 in the heavy chain of the first antibody with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)), and substitution of amino acid at EU numbering 356 in the heavy chain of the second antibody with a positively charged amino acid (e.g., lysine (K), arginine (R), or histidine (H)).

[0119] The technique of promoting heterodimerization of heavy chains by substituting lysine (K) for amino acid at EU numbering 356 in one heavy chain of an antibody and glutamic acid (E) for amino acid at EU numbering 439 in the other heavy chain is known as ART-S3 (Koga et al., MABS, Vol. 15, No. 1, 2222441, 2023). In the present invention, an antibody having lysine (K) at EU numbering 356 in one heavy chain and glutamic acid (E) at amino acid at EU numbering 439 in the other heavy chain is referred to as an ART-S3 antibody, and the substitution of lysine (K) for amino acid at EU numbering 356 in one heavy chain and glutamic acid (E) for amino acid at EU numbering 439 in the other heavy chain is referred to as an ART-S3 substitution. ART-S3 antibodies also include those with additional amino acid modifications at other positions. See also the following documents: (WO2007 / 114325; WO2006 / 106905).

[0120] Furthermore, the technique of promoting heavy chain heteropolymerization by substituting the amino acid at EU numbering 409 in one heavy chain of an antibody with lysine (K) and the amino acid at EU numbering 399 in the other heavy chain with aspartic acid (D) is called ART-S1, and this mutation is called the ART-S1 mutation, and an antibody having this modification is called the ART-S1 antibody. The technique of promoting heavy chain heteropolymerization by substituting the amino acid at EU numbering 370 in one heavy chain of an antibody with lysine (K) and the amino acid at EU numbering 357 in the other heavy chain with glutamic acid (E) is called ART-S2, and this mutation is called the ART-S2 mutation, and an antibody having this modification is called the ART-S2 antibody. The ART-S1 to ART-S3 mutations can be combined in any way, for example, by combining ART-S1 and ART-S2, ART-S1 and ART-S3, or ART-S2 and ART-S3. When combining heavy chains, it is preferable that the amino acid residues at the corresponding positions in each heavy chain be amino acids with the same charge; for example, it is preferable that all mutation positions in one heavy chain be positively charged amino acids and all mutation positions in the other heavy chain be negatively charged amino acids.

[0121] In the combination of ART-S1 and ART-S2, one heavy chain has aspartic acid (409D) at position 409 (EU numbering) and glutamic acid (370E) at position 370 (EU numbering), and the other heavy chain has 399E and 357K, respectively, which is referred to as ART-W1; in the combination of ART-S1 and ART-S3, one heavy chain has 409D and 439E, and the other heavy chain has 399K and 356K, which is referred to as ART-W2; and in the combination of ART-S2 and ART-S3, one heavy chain has 370K and 439E, and the other heavy chain has 357K and 356K, which is referred to as ART-W3 (see also Table 1 of WO2006 / 106905).

[0122] The above-described technology for promoting heavy chain heteropolymerization by introducing charged amino acids into the heavy chain constant region is part of a technology known as ART-Ig (registered trademark) (Saito, Mikiyoshi, Folia Pharmacol. Jpn., 147, 168-174 (2016)). It is not necessary to use the entire ART-Ig (registered trademark) technology; only the part of the technology that promotes heavy chain heteropolymerization can be used in this way.

[0123] In another embodiment of the present invention, the first H chain CH3 region and the second H chain CH3 region of the antibody may be cross-linked by a disulfide bond. For example, the above-mentioned ART-S1 to ART-S3 mutations can be combined with the above-mentioned 349C / 354C mutation. For example, a mutation in which one heavy chain has 409D and 349C and the other heavy chain has 399K and 354C is called ART-s1C; a mutation in which one heavy chain has 370E and 349C and the other heavy chain has 357K and 354C is called ART-s2C; and a mutation in which one heavy chain has 439E and 349C and the other heavy chain has 356K and 354C is called ART-s3C. Furthermore, a variant in which one heavy chain has 370E, 439E, and 349C and the other heavy chain has 357K, 356K, and 354C is called ART-w3C, and a variant in which one heavy chain has 370E, 439E, and 354C and the other heavy chain has 357K, 356K, and 349C is called ART-w3C2 (see also Table 1 in WO2006 / 106905). In addition, any combination of the above mutations can be used.

[0124] In addition, other known techniques can also be used to generate antibodies of the present invention. A strand-exchange engineered domain CH3 can be generated by substituting a portion of one antibody H chain CH3 with a corresponding IgA-derived sequence and then introducing the corresponding IgA-derived sequence into the complementary portion of the other H chain CH3. This allows efficient induction of association between polypeptides with different sequences through complementary association of CH3s (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technique can also be used to efficiently generate antigen-binding molecules of interest.

[0125] In addition, antibody formation may involve antibody production techniques utilizing the association of antibody CH1 and CL and VH and VL, as described in WO2011 / 028952, WO2014 / 018572, and Nat Biotechnol. 2014 Feb;32(2):191-8; techniques for producing bispecific antibodies by combining separately prepared monoclonal antibodies (Fab Arm Exchange), as described in WO2008 / 119353 and WO2011 / 131746; techniques for controlling the association between antibody heavy chain CH3s, as described in WO2012 / 058768 and WO2013 / 063702; techniques for producing antigen-binding molecules composed of two types of light chains and one type of heavy chain, as described in WO2012 / 023053; and techniques such as those described by Christoph et al. (Nature Biotechnology Vol. 31, p 753-758 (2013)).

[0126] Furthermore, pairing of the first heavy chain and the second heavy chain can be promoted by replacing the pair of CH3 of the first heavy chain and CH3 of the second heavy chain with the pair of CH1 and CL of an antibody arm (WO 09 / 80254).

[0127] These techniques disclosed herein or known in the art can be used in combination, for example, two or more of them. Furthermore, the antibodies of the present invention may be prepared based on antibodies that have been modified using these techniques.

[0128] In another preferred embodiment, the antibody of the present invention has a regulated heavy-light chain association. For example, by changing the amino acid residues at given positions in the constant region (CH1) and the constant region (CL) of the light chain of an undesired heavy-light chain combination to amino acid residues that are electrically repulsive (i.e., have the same charge), the formation of an undesired heavy-light chain combination can be prevented by utilizing this charge repulsion, resulting in the formation of a desired heavy-light chain combination.

[0129] The combination of this technique, which utilizes charge repulsion between the heavy chain constant region (CH1) and the light chain constant region (CL) to suppress undesired heavy-light chain pairing and the technique described above to promote heteropolymerization of heavy chains, is called FAST-Ig (registered trademark) (Four-chain Assembly by electrostatic steering technology - immunoglobulin) (Koga et al., MABS, vol. 15, No. 1, 2222441, 2023, doi:10.1080 / 19420862.2023.2222441). This technique enables the preferential production of desired antibodies in which the four antibody chains are properly paired. The technique for promoting heteropolymerization of heavy chains may be a steric method such as the knob-into-hole method described above, or a method based on charge repulsion or charge attraction (e.g., a method utilizing part of the ART-Ig (registered trademark) technology described above). In the present invention, mutations introduced for the FAST-Ig (registered trademark) technology are referred to as FAST-Ig (registered trademark) mutations, and antibodies developed using the FAST-Ig (registered trademark) technology are referred to as FAST-Ig (registered trademark) antibodies. A FAST-Ig (registered trademark) antibody must contain at least a FAST-Ig (registered trademark) mutation and may also contain other mutations.

[0130] The amino acid residues that come into close proximity during association can be determined, for example, by analyzing the three-dimensional structure of the polypeptide and examining the amino acid sequence of the polypeptide region that forms an interface during polypeptide association. The amino acid residues that come into close proximity at the interface are preferred targets for "modification" in the antibodies of the present invention, and any one or more of these amino acid residues can be modified.

[0131] Some amino acids are known to be charged. Generally, lysine (K), arginine (R), and histidine (H) are known to be positively charged amino acids. Aspartic acid (D), glutamic acid (E), and the like are known to be negatively charged amino acids. In addition, alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), and the like are known to be uncharged or nonpolar amino acids.

[0132] Therefore, amino acids that are electrically repelled from one another (have the same charge) in the present invention refer to the following: (1) amino acids in which one of the amino acids is a positively charged amino acid and the other amino acid is also a positively charged amino acid, and (2) amino acids in which one of the amino acids is a negatively charged amino acid and the other amino acid is also a negatively charged amino acid.

[0133] The amino acid modification in the present invention includes multiple modifications to positively charged amino acids on either the heavy chain or the light chain, and multiple modifications to negatively charged amino acids on the other chain.Furthermore, multiple modifications to positively charged amino acids and multiple modifications to negatively charged amino acids can be made on the same heavy chain or light chain.In these modifications, modifications to uncharged or nonpolar amino acids and modifications to uncharged or nonpolar amino acids can also be suitably combined.

[0134] In the modifications of the present invention, for example, amino acids on one of the chains can be used unmodified, in which case both the heavy and light chains need not be modified, but only one of the chains may be modified.

[0135] The light chain constant region of the antibody of the present invention is preferably a human light chain constant region. Examples of antibody light chain constant regions include IgK(κ), IgL1, IgL2, IgL3, IgL6, and IgL7(λ) constant regions. The light chain constant region of the antibody of the present invention is not particularly limited; when multiple types of light chains are used, the light chains may be of different types, such as κ and λ. Several allotype sequences obtained by genetic polymorphism are described in Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, as human IgK(κ) constant regions and human IgL7(λ) constant regions, and any of these may be used in the present invention.

[0136] The antibodies of the present invention include heterotetramers having two types of CH1 and two types of CL. The heterotetramers preferably have bivalent or more binding sites, and the bivalent binding sites may bind to the same epitope, but preferably bind to at least two different epitopes, such as multispecific antibodies.

[0137] A preferred example of the multispecific antibody of the present invention is a bispecific antibody. An example of a preferred embodiment of the antibody of the present invention is a bispecific antibody composed of two types of heavy chains (a first heavy chain and a second heavy chain) and two types of light chains (a first light chain and a second light chain).

[0138] To more precisely describe the "bispecific antibody" of a preferred embodiment of the antibody of the present invention, the above-mentioned "first heavy chain" refers to one of the two heavy chains (H chains) that form the antibody, and the "second H chain" refers to the other H chain that is different from the first H chain. That is, one of the two H chains can be arbitrarily defined as the first H chain, and the other can be defined as the second H chain. The "first light chain" refers to one of the two light chains (L chains) that form the bispecific antibody, and the "second L chain" refers to the other L chain that is different from the first L chain. Of the two L chains, the L chain that exhibits antigen-binding activity by pairing with the first H chain is usually referred to as the "first light chain," and the L chain that exhibits antigen-binding activity by pairing with the second H chain is usually referred to as the "second light chain." Typically, the first L chain and the first H chain are derived from the same antibody that binds to a specific antigen (or epitope), and the second L chain and the second H chain are also derived from the same antibody that binds to a specific antigen (or epitope). Herein, the L chain-H chain pair formed by the first H chain and the first L chain is referred to as the first pair, and the L chain-H chain pair formed by the second H chain and the second L chain is referred to as the second pair. The H chains of the first pair and the H chains of the second pair have different amino acid sequences. The epitope (preferably the antigen) used to generate the antibody from which the second pair is derived may be the same as, but preferably is different from, the epitope (preferably the antigen) used to generate the antibody from which the first pair is derived. More specifically, the antigens recognized by the first pair and the second pair may be the same, but preferably the pairs bind to different epitopes (preferably the antigens). Furthermore, the L chains of the first pair and the L chains of the second pair have different amino acid sequences. When the first pair and the second pair bind to different epitopes, they may recognize completely different antigens or different sites (different epitopes) on the same antigen. Furthermore, one of them may recognize an antigen such as a protein, peptide, gene, or sugar, while the other may recognize a cytotoxic substance such as a radioactive substance, a chemotherapeutic agent, or a cell-derived toxin.However, if it is desired to produce antibodies having pairs formed by a specific combination of H chains and L chains, those specific H chains and L chains may be arbitrarily determined to be the first pair and the second pair.

[0139] A more detailed description is provided below for the case of an IgG-type bispecific antibody having two types of heavy chain constant regions CH1 (CH1-A and CH1-B) and two types of light chain constant regions (CL-A and CL-B); however, the present invention can be applied to other antibodies as well.

[0140] For example, the amino acid residues forming the interface between the undesired pair CH1-A and CL-B are modified to positively charged amino acid residues, and the amino acid residues forming the interface between the undesired pair CH1-B and CL-A are modified to negatively charged amino acid residues. As a result of these modifications, the unintended association between CH1-A and CL-B is inhibited because both amino acid residues forming the interface are positively charged, and the association between CH1-B and CL-A is also inhibited because both amino acid residues forming the interface are negatively charged. In this way, the unintended association between CH1-A and CL-B and between CH1-B and CL-A are inhibited because the amino acid residues forming the interface have the same charge. As a result, antibodies having the intended association between CH1-A and CL-A and the intended association between CH1-B and CL-B can be efficiently obtained. Furthermore, the intended association between CH1-A and CL-A is promoted because the amino acid residues forming the interface have different types of charges; and the intended association between CH1-B and CL-B is also promoted because the amino acid residues forming the interface have different types of charges. As a result, antibodies with the intended association can be efficiently obtained.

[0141] In another example, when the amino acid residues forming the interface between CL-A and CH1-B are mutually uncharged or nonpolar amino acids, the amino acid residues forming the interface between CH1-A and CL-B are modified to positively charged amino acid residues. As a result of this modification, unintended association between CH1-A and CL-B is inhibited because both amino acid residues forming the interface are positively charged. On the other hand, because the amino acid residues forming the interface are not mutually electrically repulsive, the intended association between CH1-A and CL-A and between CH1-B and CL-B is thought to occur more easily than when the amino acids are electrically repulsive. As a result, antibodies having the intended association between CH1-A and CL-A and between CH1-B and CL-B can be efficiently obtained. On the other hand, in this example, when the amino acid residues forming the interface between CL-A and CH1-B are not mutually uncharged or nonpolar amino acids, they can be modified to become mutually uncharged or nonpolar amino acids.

[0142] In another example, when the amino acid residues forming the interface between CL-B and CH1-B are uncharged or nonpolar in CH1-B, one of the amino acid residues forming the interface between CH1-A and CL-A is modified to a positively charged amino acid residue, while the other is modified to a negatively charged amino acid residue; and the amino acid residues forming the interface between CL-B and CH1-B in CL-B are modified to have the same charge as the modification made to CH1-A. As a result of this modification, the intended association between CH1-A and CL-A is promoted because the amino acid residues forming the interface have a combination of positive and negative charges, while the intended association between CH1-B and CL-B is not inhibited because the amino acid residues forming the interface are amino acids that do not electrically repel each other. As a result, antibodies having the intended association between CH1-A and CL-A and the intended association between CH1-B and CL-B can be efficiently obtained. On the other hand, in this example, if the amino acid residues forming the interface between CL-B and CH1-B are not uncharged or nonpolar amino acids in CH1-B, they may be modified to become uncharged or nonpolar amino acids.

[0143] In addition, the use of the association modulation of the present invention makes it possible to inhibit the association between CH1 (CH1-A and CH1-B) or the association between CL (CL-A and CL-B).

[0144] Those skilled in the art will be able to suitably determine the types of amino acid residues that are accessed during association at the CH1 and CL interface in a desired polypeptide for which modulation of association according to the present invention is desired.

[0145] Furthermore, those skilled in the art can also suitably obtain sequences that can be used as CH1 or CL of antibodies in organisms such as humans, monkeys, mice, rabbits, etc., by using public databases, etc.

[0146] Examples of such antibodies include those in which a first heavy chain and a first light chain are paired and a second heavy chain and a second light chain are paired, such as the following: (1) An antibody in which the amino acid at position 147 and / or 175 (EU numbering) in the constant region of the first heavy chain (CH1) and the amino acid at position 131, 160, and / or 180 (EU numbering) in the light chain constant region of the second light chain (CL) are mutually electrically repulsive amino acid residues. (2) An antibody in which the amino acid at position 213 (EU numbering) in the constant region of the first heavy chain (CH1) and the amino acid at position 123 (EU numbering) in the light chain constant region of the second light chain (CL) are mutually electrically repulsive amino acid residues.

[0147] For example, in the above (1), the antibody has amino acid residues that are electrically repulsive between either or both of positions 147 and 175 (EU numbering) in the first heavy chain and either or any combination of positions 131, 160, and 180 (EU numbering) in the second light chain. This inhibits pairing between the first heavy chain and the second light chain. In the above (2), the antibody has amino acid residues that are electrically repulsive between the amino acid residue at position 213 (EU numbering) in the first heavy chain and the amino acid residue at position 123 (EU numbering) in the second light chain. This inhibits pairing between the first heavy chain and the second light chain. The above (1) and (2) may be combined, in which either or both of the amino acid residues at positions 147 and 175 (EU numbering) in the first heavy chain and either or any combination of the amino acid residues at positions 131, 160, and 180 (EU numbering) in the second light chain are mutually electrically repulsive amino acid residues, and the amino acid residue at position 213 (EU numbering) in the first heavy chain and the amino acid residue at position 123 (EU numbering) in the second light chain are mutually electrically repulsive amino acid residues. Such an embodiment is encompassed by both (1) and (2) above.

[0148] The amino acid residues in the second heavy chain corresponding to the positions of the electrically repulsive amino acid residues in the first heavy chain are non-electrically repulsive amino acid residues, i.e., uncharged amino acid residues or electrically attractive amino acid residues. Furthermore, the amino acid residues in the first light chain corresponding to the positions of the electrically repulsive amino acid residues in the second light chain are non-electrically repulsive amino acid residues, i.e., uncharged amino acid residues or electrically attractive amino acid residues. This suppresses undesired pairings between heavy and light chains and allows the desired pairings to be preferentially formed.

[0149] Electrically repulsive amino acid residues include positively charged amino acids such as lysine (K), arginine (R), and histidine (H), which are electrically repulsive to each other. Negatively charged amino acids include glutamic acid (E) and aspartic acid (D), which are electrically repulsive to each other. When producing the antibodies of the present invention, these amino acids can be substituted as appropriate to introduce a charge, with lysine (K) and glutamic acid (E) being particularly preferred as the substituted amino acid.

[0150] For example, for bispecific antibodies, specific examples of amino acid residues that approach (face or contact) at the interface between CH1 and CL upon association include the following combinations: - glutamine (Q) at position 175 according to EU numbering in CH1 and glutamine (Q) or glutamic acid (E) at position 160 according to Kabat numbering in the facing (contacting) CL; - glutamine (Q) at position 175 according to EU numbering in CH1 and threonine (T) or serine (S) at position 131 according to Kabat numbering in the facing (contacting) CL; - glutamine (Q) at position 175 according to EU numbering in CH1 and serine (S) or threonine (T) at position 131 according to Kabat numbering and glutamine (Q) or glutamic acid (E) at position 160 according to Kabat numbering in the facing (contacting) CL; and - Lysine (K) at position 147 and glutamine (Q) at position 175 according to EU numbering in CH1, and serine (S) or threonine (T) at position 131 and glutamine (Q) or glutamic acid (E) at position 160 according to Kabat numbering in the opposing (contacting) CL.

[0151] In the present invention, numbers written in EU numbering are indicated according to EU numbering (Sequences of proteins of immunological interest, NIH Publication No. 91-3242). In the present invention, the phrases "amino acid residue at position X according to EU numbering" and "amino acid at position X according to EU numbering" (X is any number) can also be read as "amino acid residue corresponding to position X according to EU numbering" and "amino acid corresponding to position X according to EU numbering." Desired antibodies can be obtained by modifying these amino acid residues.

[0152] In one aspect, the present invention provides an antibody in which the association of heavy and light chains is regulated, wherein one or two or more sets of amino acid residues selected from the group consisting of the following sets of amino acid residues (a) to (j) in the heavy and light chains of the antibody are amino acid residues that are electrically repulsive to each other: (a) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residue at position 160 in CL according to Kabat numbering; (b) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residue at position 131 in CL according to Kabat numbering; (c) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residue at position 180 in CL according to Kabat numbering; (d) the amino acid residue at position 147 in CH1 according to EU numbering and the amino acid residue at position 131 in CL according to Kabat numbering; (e) (f) the amino acid residue in CH1 at position 147 according to EU numbering and the amino acid residue in CL at position 160 according to Kabat numbering; (g) the amino acid residue in CH1 at position 213 according to EU numbering and the amino acid residue in CL at position 123 according to Kabat numbering; (h) the amino acid residues in CH1 at positions 147 and 175 according to EU numbering and the amino acid residues in CL at positions 131 and 160 according to Kabat numbering; (i) the amino acid residues in CH1 at positions 147 and 175 according to EU numbering and the amino acid residues in CL at positions 131 and 180 according to Kabat numbering; and (j) The amino acid residue contained in CH1 at position 175 according to EU numbering, and the amino acid residues contained in CL at positions 131 and 160 according to Kabat numbering.

[0153] In the above-described antibodies, the "mutually electrically repulsive amino acid residues" or "amino acid residues having the same charge" are preferably selected from amino acid residues contained in either of the following sets (X) or (Y): (X) glutamic acid (E) or aspartic acid (D); or (Y) lysine (K), arginine (R), or histidine (H).

[0154] In the above-mentioned antibodies, specific examples of sets of amino acid residues that are electrically repulsive to each other include the following sets of amino acid residues: (a) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residue at position 160 in CL according to EU numbering; (b) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residue at position 131 in CL according to Kabat numbering; (c) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residue at position 180 in CL according to Kabat numbering; (d) the amino acid residue at position 147 in CH1 according to EU numbering and the amino acid residue at position 131 in CL according to Kabat numbering; (e) the amino acid residue at position 147 in CH1 according to EU numbering and the amino acid residue at position 160 in CL according to Kabat numbering; (f) (g) the amino acid residue at position 213 in CH1 according to EU numbering and the amino acid residue at position 123 in CL according to Kabat numbering; (h) the amino acid residues at positions 147 and 175 in CH1 according to EU numbering and the amino acid residues at positions 131 and 160 in CL according to Kabat numbering; (i) the amino acid residues at positions 147 and 175 in CH1 according to EU numbering and the amino acid residues at positions 131 and 180 in CL according to Kabat numbering; (j) the amino acid residue at position 175 in CH1 according to EU numbering and the amino acid residues at positions 131 and 160 in CL according to Kabat numbering.

[0155] In some embodiments, in the antibody of the present invention, one, two, three, or all of the sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in the following (a) to (j) in the heavy chain and light chain of the antibody are amino acid residues that are electrostatically repulsive to each other: (a) an amino acid residue in the heavy chain constant region (CH1) that is at position 175 according to EU numbering and an amino acid residue in the light chain constant region (CL) that is at position 131 according to Kabat numbering, (b) an amino acid residue in CH1 that is at position 175 according to EU numbering and an amino acid residue in CL that is at position 160 according to Kabat numbering, (c) an amino acid residue in CH1 that is at position 175 according to EU numbering and an amino acid residue in CL that is at position 180 according to Kabat numbering, (d) an amino acid residue in CH1 that is at position 147 according to EU numbering and an amino acid residue in CL that is at position 131 according to Kabat numbering; (e) (f) the amino acid residue in CH1 which is at position 147 according to EU numbering and the amino acid residue in CL which is at position 160 according to Kabat numbering; (g) the amino acid residue in CH1 which is at position 213 according to EU numbering and the amino acid residue in CL which is at position 123 according to Kabat numbering; (h) the amino acid residue in CH1 which is at position 175 according to EU numbering and the amino acid residues in CL which are at positions 131 and 160 according to Kabat numbering; (i) the amino acid residues in CH1 which are at positions 147 and 175 according to EU numbering and the amino acid residues in CL which are at positions 131 and 180 according to Kabat numbering; (j) The amino acid residues in CH1 are positions 147 and 175 according to EU numbering, and the amino acid residues in CL are positions 131 and 160 according to Kabat numbering.

[0156] The present invention provides antibodies, wherein one or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a1) to (j2) below in the heavy chain and light chain of the antibody are amino acid residues that are electrically repulsive to each other: (a1) the amino acid residue at position 175 in CH1 according to EU numbering, which is glutamic acid (E) or aspartic acid (D), and the amino acid residue at position 160 in CL according to EU numbering, which is glutamic acid (E) or aspartic acid (D); (a2) the amino acid residue at position 175 in CH1 according to EU numbering, which is lysine (K), histidine (H), or arginine (R), and the amino acid residue at position 160 in CL according to EU numbering, which is lysine (K), histidine (H), or arginine (R); (b1) (b2) the amino acid residue in CH1 at position 175 according to EU numbering is lysine (K), histidine (H), or arginine (R), and the amino acid residue in CL at position 131 according to EU numbering is lysine (K), histidine (H), or arginine (R); (c1) the amino acid residue in CH1 at position 175 according to EU numbering is glutamic acid (E) or aspartic acid (D), and the amino acid residue in CL at position 180 according to EU numbering is glutamic acid (E) or aspartic acid (D); (c2) an amino acid residue contained in CH1 at position 175 according to EU numbering, which is lysine (K), histidine (H), or arginine (R), and an amino acid residue contained in CL at position 180 according to EU numbering, which is lysine (K), histidine (H), or arginine (R);(d1) the amino acid residue in CH1 at position 147 according to EU numbering is glutamic acid (E) or aspartic acid (D), and the amino acid residue in CL at position 131 according to EU numbering is glutamic acid (E) or aspartic acid (D); (d2) the amino acid residue in CH1 at position 147 according to EU numbering is lysine (K), histidine (H), or arginine (R), and the amino acid residue in CL at position 131 according to EU numbering is lysine (K), histidine (H), or arginine (R); (e1) the amino acid residue in CH1 at position 147 according to EU numbering is glutamic acid (E) or aspartic acid (D), and the amino acid residue in CL at position 160 according to EU numbering is glutamic acid (E) or aspartic acid (D); (e2) (f1) the amino acid residue in CH1 at position 147 according to EU numbering is lysine (K), histidine (H), or arginine (R), and the amino acid residue in CL at position 160 according to EU numbering is lysine (K), histidine (H), or arginine (R); (f2) the amino acid residue in CH1 at position 147 according to EU numbering is lysine (K), histidine (H), or arginine (R), and the amino acid residue in CL at position 180 according to EU numbering is glutamic acid (E) or aspartic acid (D); (g1) an amino acid residue contained in CH1 at position 213 according to EU numbering, which is glutamic acid (E) or aspartic acid (D), and an amino acid residue contained in CL at position 123 according to EU numbering, which is glutamic acid (E) or aspartic acid (D);(g2) the amino acid residue in CH1 at position 213 according to EU numbering, which is lysine (K), histidine (H), or arginine (R), and the amino acid residue in CL at position 123 according to EU numbering, which is lysine (K), histidine (H), or arginine (R); (h1) the amino acid residues in CH1 at positions 147 and 175 according to EU numbering, which are glutamic acid (E) or aspartic acid (D), respectively, and the amino acid residues in CL at positions 131 and 160 according to EU numbering, which are glutamic acid (E) or aspartic acid (D), respectively; (h2) (i1) the amino acid residues contained in CH1 at positions 147 and 175 according to EU numbering, which are lysine (K), histidine (H), or arginine (R), respectively, and the amino acid residues contained in CL at positions 131 and 160 according to EU numbering, which are lysine (K), histidine (H), or arginine (R), respectively; (i2) the amino acid residues contained in CH1 at positions 147 and 175 according to EU numbering, which are glutamic acid (E) or aspartic acid (D), respectively, and the amino acid residues contained in CL at positions 131 and 180 according to EU numbering, which are glutamic acid (E) or aspartic acid (D), respectively; (i2) (j1) the amino acid residues contained in CH1 at positions 147 and 175 according to EU numbering, which are lysine (K), histidine (H), or arginine (R), respectively, and the amino acid residues contained in CL at positions 131 and 180 according to EU numbering, which are lysine (K), histidine (H), or arginine (R), respectively; (j1) the amino acid residue contained in CH1 at position 175 according to EU numbering, which is glutamic acid (E) or aspartic acid (D), respectively, and the amino acid residues contained in CL at positions 131 and 160 according to EU numbering, which are glutamic acid (E) or aspartic acid (D);(j2) the amino acid residue contained in CH1 at position 175 according to EU numbering, which is lysine (K), histidine (H), or arginine (R), respectively, and the amino acid residues contained in CL at positions 131 and 160 according to EU numbering, which are lysine (K), histidine (H), or arginine (R), respectively;

[0157] In the above-mentioned antibodies, specific examples of amino acid residues that are electrically repulsive to each other include the following amino acid residues: (a1) the amino acid residue at position 175 in CH1 according to EU numbering, which is glutamic acid (E) or aspartic acid (D), and the amino acid residue at position 160 in CL, which is glutamic acid (E) or aspartic acid (D); (a2) the amino acid residue at position 175 in CH1 according to EU numbering, which is lysine (K), histidine (H), or arginine (R), and the amino acid residue at position 160 in CL, which is lysine (K), histidine (H), or arginine (R); (b1) (b2) the amino acid residue in CH1 at position 175 according to EU numbering is lysine (K), histidine (H), or arginine (R), and the amino acid residue in CL at position 131 according to EU numbering is lysine (K), histidine (H), or arginine (R); (c1) the amino acid residue in CH1 at position 175 according to EU numbering is glutamic acid (E) or aspartic acid (D), and the amino acid residue in CL at position 180 according to EU numbering is glutamic acid (E) or aspartic acid (D); (c2) (d1) the amino acid residue in CH1 at position 175 according to EU numbering, which is lysine (K), histidine (H), or arginine (R), and the amino acid residue in CL at position 180 according to EU numbering, which is lysine (K), histidine (H), or arginine (R); (d2) the amino acid residue in CH1 at position 147 according to EU numbering, which is glutamic acid (E) or aspartic acid (D), and the amino acid residue in CL at position 131 according to EU numbering, which is glutamic acid (E) or aspartic acid (D);(d2) the amino acid residue at position 147 in CH1 according to EU numbering is lysine (K), histidine (H), or arginine (R), and the amino acid residue at position 131 in CL according to EU numbering is lysine (K), histidine (H), or arginine (R); (e1) the amino acid residue at position 147 in CH1 according to EU numbering is glutamic acid (E) or aspartic acid (D), and the amino acid residue at position 160 in CL according to EU numbering is glutamic acid (E) or aspartic acid (D); (e2) the amino acid residue at position 147 in CH1 according to EU numbering is lysine (K), histidine (H), or arginine (R), and the amino acid residue at position 160 in CL according to EU numbering is lysine (K), histidine (H), or arginine (R); (f1) the amino acid residue in CH1 at position 147 according to EU numbering, which is glutamic acid (E) or aspartic acid (D), and the amino acid residue in CL at position 180 according to EU numbering, which is glutamic acid (E) or aspartic acid (D); (f2) the amino acid residue in CH1 at position 147 according to EU numbering, which is lysine (K), histidine (H), or arginine (R), and the amino acid residue in CL at position 180 according to EU numbering, which is lysine (K), histidine (H), or arginine (R); (g1) the amino acid residue in CH1 at position 213 according to EU numbering, which is glutamic acid (E) or aspartic acid (D), and the amino acid residue in CL at position 123 according to EU numbering, which is glutamic acid (E) or aspartic acid (D); (g2) an amino acid residue contained in CH1 at position 213 according to EU numbering, which is lysine (K), histidine (H), or arginine (R), and an amino acid residue contained in CL at position 123 according to EU numbering, which is lysine (K), histidine (H), or arginine (R);(h1) the amino acid residues in CH1 at positions 147 and 175 according to EU numbering are glutamic acid (E) or aspartic acid (D), respectively, and the amino acid residues in CL at positions 131 and 160 according to EU numbering are glutamic acid (E) or aspartic acid (D), respectively; (h2) the amino acid residues in CH1 at positions 147 and 175 according to EU numbering are lysine (K), histidine (H), or arginine (R), respectively, and the amino acid residues in CL at positions 131 and 160 according to EU numbering are lysine (K), histidine (H), or arginine (R); (i1) (i1) the amino acid residues in CH1 at positions 147 and 175 according to EU numbering, which are glutamic acid (E) or aspartic acid (D), respectively, and the amino acid residues in CL at positions 131 and 180 according to EU numbering, which are glutamic acid (E) or aspartic acid (D), respectively; (i2) the amino acid residues in CH1 at positions 147 and 175 according to EU numbering, which are lysine (K), histidine (H), or arginine (R), respectively, and the amino acid residues in CL at positions 131 and 180 according to EU numbering, which are lysine (K), histidine (H), or arginine (R); (j1) (j2) the amino acid residue in CH1 at position 175 according to EU numbering is glutamic acid (E) or aspartic acid (D), and the amino acid residues in CL at positions 131 and 160 according to EU numbering are glutamic acid (E) or aspartic acid (D), respectively; (j3) the amino acid residue in CH1 at position 175 according to EU numbering is lysine (K), histidine (H), or arginine (R), and the amino acid residues in CL at positions 131 and 160 according to EU numbering are lysine (K), histidine (H), or arginine (R), respectively;

[0158] In addition to the above, strategies can be further applied to inhibit undesired CH1 / CL association by introducing charge repulsion at the interface between CH1 and CL (WO 2013 / 065708; WO2019 / 065795).

[0159] In another embodiment, the antibody of the present invention may have the amino acids at positions 123 and / or 124 in the light chain constant domain CL of each of the first and second antigen-binding moieties independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering). In addition, or in another aspect, the antibody of the present invention may have the amino acids at positions 147 and / or 213 in the heavy chain constant domain CH1 of each of the first and second antigen-binding moieties independently substituted with glutamic acid (E) or aspartic acid (D) (EU numbering).

[0160] In another embodiment, the antibody of the present disclosure can have the amino acids at positions 123 and 124 in the constant domain CL of the light chain of each of the first and second antigen-binding moieties be arginine (R) and lysine (K), respectively (Kabat numbering). In addition, or in another aspect, the antibody of the present disclosure can have the amino acids at positions 147 and 213 in the constant domain CH1 of the heavy chain of each of the first and second antigen-binding moieties be glutamic acid (E) (EU numbering).

[0161] In the above-mentioned embodiment, the constant region is preferably of human origin. In a particularly preferred embodiment, the subclass of the CL region is κ or λ.

[0162] In addition, in a method for introducing electrical repulsion into the interface between the heavy and light chain variable regions to suppress undesired association of the heavy and light chains, examples of amino acid residues that contact at the interface between the heavy and light chain variable regions (VH and VL) include glutamine (Q) at position 39 according to the Kabat numbering in VH (FR2 region) and glutamine (Q) at position 38 according to the Kabat numbering in the opposing (contacting) VL (FR2 region). Further preferred examples are leucine (L) at position 45 according to the Kabat numbering in VH (FR2) and proline (P) at position 44 according to the Kabat numbering in the opposing VL (FR2).

[0163] These amino acid residues are known to be highly conserved in humans and mice (J. Mol. Recognit. 2003; 16: 113-120), and therefore, the association of the antibody variable region can be adjusted by modifying the amino acid residues corresponding to the above-mentioned amino acid residues.

[0164] In some embodiments, in the antibodies of the present invention, two or more amino acid residues forming the interface between the heavy chain variable region and the light chain variable region are amino acid residues that are electrostatically repulsive to each other.

[0165] A specific example is an antibody in which two or more amino acid residues forming the interface between VH and VL are mutually electrically repulsive amino acid residues. More specifically, examples include antibodies having one or two amino acid residue sets selected from the group consisting of the amino acid residue sets shown in (a) or (b) below: (a) the amino acid residue at position 39 in VH according to Kabat numbering and the amino acid residue at position 38 in VL according to Kabat numbering; or (b) the amino acid residue at position 45 in VH according to Kabat numbering and the amino acid residue at position 44 in VL according to Kabat numbering.

[0166] In some embodiments, in the antibodies of the present invention, the amino acid residues that are electrostatically repulsive to each other are one or two sets of amino acid residues selected from the group consisting of the following sets of amino acid residues (a) and (b): (a) an amino acid residue in the heavy chain variable region that is at position 39 according to the Kabat numbering system and an amino acid residue in the light chain variable region that is at position 38 according to the Kabat numbering system; (b) an amino acid residue in the heavy chain variable region that is at position 45 according to the Kabat numbering system and an amino acid residue in the light chain variable region that is at position 44 according to the Kabat numbering system.

[0167] The amino acid residues described in (a) or (b) above are close to each other during assembly. Those skilled in the art will be able to find positions in the desired VH or VL corresponding to the amino acid residues described in (a) or (b) above by homology modeling using commercially available software, and appropriately modify the amino acid residues at those positions.

[0168] In some embodiments, in the multispecific antibody, the mutually electrostatically repulsive amino acid residues are selected from amino acid residues included in either the following set (X) or (Y): (X) glutamic acid (E), aspartic acid (D), (Y) lysine (K), arginine (R), histidine (H).

[0169] In a method for modifying amino acid residues forming the hydrophobic core at the interface between VH and VL to charge polar amino acids so as to suppress unintended association between the heavy and light chains, preferred examples of amino acid residues capable of forming the hydrophobic core at the interface between VH and VL include leucine (L) at position 45 according to the Kabat numbering in VH (FR2) and proline (P) at position 44 according to the Kabat numbering in the opposing VL (FR2).

[0170] Generally, the term "hydrophobic core" refers to the portion formed by the collection of hydrophobic amino acid side chains inside an assembled polypeptide. Examples of hydrophobic amino acids include alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In addition, amino acid residues other than hydrophobic amino acids (such as tyrosine) can participate in the formation of the hydrophobic core. This hydrophobic core, together with the hydrophilic surface where the hydrophilic amino acid side chains are exposed to the outside, becomes the driving force for promoting the association of water-soluble polypeptides. When the hydrophobic amino acids of two different domains are present on the molecular surface and exposed to water molecules, the entropy will increase and the free energy will increase. Therefore, the two domains will associate with each other to reduce free energy and become stable, and the hydrophobic amino acids at the interface will be buried in the interior of the molecule to form a hydrophobic core.

[0171] When polypeptide association occurs, modifying the hydrophobic amino acids that form the hydrophobic core to polar, charged amino acids is believed to inhibit the formation of the hydrophobic core; consequently, peptide association is believed to be inhibited.

[0172] Those skilled in the art can recognize the presence or absence of a hydrophobic core, as well as the site (region) where it is formed, by analyzing the amino acid sequence of a desired antibody. That is, the antibodies of the present invention are characterized in that amino acid residues that can form a hydrophobic core at the interface have been modified to have charged amino acid residues. More specifically, examples include antibodies in which the amino acid residues shown in either (1) or (2) below are charged amino acid residues. The side chains of the amino acid residues shown in (1) and (2) below are adjacent to each other and can form a hydrophobic core: (1) the amino acid residue at position 45 in VH according to the Kabat numbering system; and (2) the amino acid residue at position 44 in VL according to the Kabat numbering system.

[0173] Preferred examples of charged amino acid residues in the antibodies include glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), and histidine (H). More preferred examples include glutamic acid (E) and lysine (K).

[0174] Generally, the amino acid residues described in (1) and (2) above in humans and mice are respectively: (1) leucine (L), and (2) proline (P). Therefore, in a preferred embodiment of the present invention, these amino acid residues are subjected to modification (e.g., substitution with an amino acid having a charge). Furthermore, the types of amino acid residues described in (1) and (2) above are not necessarily limited to the aforementioned amino acid residues, but may be other amino acids equivalent to these amino acid residues.

[0175] Other known techniques can be applied to the antibodies of the present invention. For example, to promote the association between a first VH (VH1) and a first VL (VL1) and / or a second VH (VH2) and a second VL (VL2), an amino acid side chain in one variable region of the heavy chain can be replaced with a larger side chain (knob), and an amino acid side chain in the opposing variable region of the other heavy chain can be replaced with a smaller side chain (hole), thereby positioning the knob in the hole, promoting the association between VH1 and VL1 and / or VH2 and VL2; and consequently, further suppressing the association between VH1 and VL2 and / or VH2 and VL1.

[0176] Furthermore, to facilitate the separation and purification of a target antibody in which the heavy chains of the antibody are heteropolymerized using a chromatography column or the like, modifications can be introduced that increase the difference in isoelectric point compared to antibodies in which the heavy chains are homopolymerized. This technology is part of the aforementioned technology known as ART-Ig (registered trademark) (Saito, Mikiyoshi, Folia Pharmacol. Jpn., 147, 168-174 (2016)), and modifications have been identified in the heavy chains of antibodies that can control the isoelectric point without reducing the function (activity) of the antibody. In the present invention, the part of the ART-Ig (registered trademark) technology that controls the isoelectric point can also be utilized.

[0177] For examples of modifications, reference can be made to WO2006 / 106905 and the like as appropriate. Specific examples include: in the first heavy chain of an antibody, (i) amino acid residues at positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 97, 105, 108, 110, and 112 according to the Kabat numbering in the variable region of the heavy chain; and (ii) at least one amino acid residue selected from amino acid residues at positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 (EU numbering) in the heavy chain constant region is charged; and in the second heavy chain of the antibody, (i) (ii) amino acid residues at positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 97, 105, 108, 110, and 112 according to the Kabat numbering system in the heavy chain variable region; and Antibodies include antibodies in which at least one amino acid residue selected from amino acid residues at positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 (EU numbering) in the heavy chain constant region has an opposite charge to the charged amino acid residue selected in the first heavy chain, or is uncharged. This results in a difference in the isoelectric points of the first heavy chain and the second heavy chain, resulting in a difference in the isoelectric point of the heteropolymer from those of the first heavy chain homopolymer and the second heavy chain homopolymer. This facilitates separation by chromatography (cation exchange chromatography or anion exchange chromatography) or the like, and enables increased production efficiency of bispecific antibodies and the like. Preferably, the difference in isoelectric points of the first heavy chain and the second heavy chain is at least 0.5.

[0178] In a preferred embodiment, the combination of the amino acid residue having the charge and the amino acid residue having the opposite charge to the amino acid residue is selected from amino acid residues included in either the following group (a) or (b): (a) glutamic acid (E), aspartic acid (D); (b) lysine (K), arginine (R), histidine (H).

[0179] Furthermore, when the first heavy chain and the second heavy chain have constant regions, the heavy chains may be derived from constant regions of different antibody subclasses. A difference in isoelectric point can be achieved by selecting a combination of constant regions with different isoelectric points from different subclasses and using this combination as the constant region for the first heavy chain and the second heavy chain. For example, the constant region for the first heavy chain and the constant region for the second heavy chain may be derived from IgG1 and IgG4, respectively, or from IgG1 and IgG2, respectively. Note that "first" and "second" are interchangeable, and the constant region for the first heavy chain and the constant region for the second heavy chain may be derived from IgG4 and IgG1, respectively, or from IgG2 and IgG1, respectively. The difference in isoelectric point can be increased by further introducing modifications into the above-mentioned sites in the constant regions of different subclasses.

[0180] To produce the above-described antibodies of the present invention (including polypeptides containing antigen-binding fragments thereof), the present invention uses a vector containing four coding regions encoding two types of antibody heavy chains or antigen-binding fragments thereof and two types of light chains or antigen-binding fragments thereof corresponding to each heavy chain, in a copy number ratio of 1:1:1:1 in a single vector, and a cell into which the vector has been introduced.

[0181] Specifically, the present invention provides a vector containing four foreign nucleic acids in a 1:1:1:1 ratio, each encoding two antibody heavy chains or their antigen-binding fragments, and two corresponding light chains or their antigen-binding fragments, and a cell into which the vector has been introduced. Here, the four foreign nucleic acids encoding the two antibody heavy chains or their antigen-binding fragments, and the two corresponding light chains or their antigen-binding fragments, are foreign nucleic acids that encode these four polypeptide chains. Furthermore, "foreign nucleic acid" refers to nucleic acid introduced into the cell. Nucleic acids can be introduced into cells by introducing a vector carrying the nucleic acid or the nucleic acid itself into the cell. The cell is a transformed cell into which a nucleic acid containing the above coding region has been introduced.

[0182] The present inventors have found that antibodies comprising two heavy chains and two light chains can be produced with high efficiency by using cells containing equal copy numbers of four coding regions encoding two heavy chains and two light chains of an antibody, i.e., 1:1:1:1.

[0183] For example, in the case of two types of heavy chains and two types of light chains possessed by an antibody, the four coding regions encoding four types of polypeptides, namely, a first heavy chain, a first light chain paired with it, and a second heavy chain, a second light chain paired with it, are preferably used in the present invention. For example, the following vectors contain one copy of each of the four coding regions in a single vector, and cells into which the vector has been introduced; a vector containing two copies of each of the coding regions in a single vector, and cells into which the vector has been introduced; a vector containing three copies of each of the coding regions in a single vector, and cells into which the vector has been introduced; a vector containing four copies of each of the coding regions in a single vector, and cells into which the vector has been introduced; a vector containing five copies of each of the coding regions in a single vector, and cells into which the vector has been introduced; a vector containing six copies of each of the coding regions in a single vector, and cells into which the vector has been introduced; a vector containing seven copies of each of the coding regions in a single vector, and cells into which the vector has been introduced; a vector containing eight copies of each of the coding regions in a single vector, and cells into which the vector has been introduced. Suitable examples include a vector containing two copies of each of the four coding regions encoding the four polypeptides (two heavy chains and two light chains) (i.e., a total of eight coding regions encoding antibody chains) in a single vector, and a cell into which the vector has been introduced; or a vector containing four copies of each of the four coding regions (i.e., a total of 16 coding regions encoding antibody chains) in a single vector, and a cell into which the vector has been introduced. For example, a cell into which two vectors containing two copies of each of the four coding regions encoding the four polypeptides (two heavy chains and two light chains) (i.e., a total of eight coding regions encoding antibody chains) in a single vector are preferred. Also suitable examples include a vector containing one copy of each of the four coding regions encoding the four polypeptides (two heavy chains and two light chains) (i.e., a total of four coding regions encoding antibody chains) in a single vector, and a cell into which the vector has been introduced. For example, cells into which two vectors containing one copy each of the four coding regions encoding the four polypeptides of the two heavy chains and two light chains (i.e., a total of four coding regions encoding antibody chains) have been introduced are also preferred.

[0184] The exogenous nucleic acid may be expressed in cells by transient expression or stable expression, but is preferably expressed in a stable expression system. For example, it is possible to express the exogenous nucleic acid under desired conditions using site-specific recombination techniques, inducible promoters, condition-dependent promoters, etc.

[0185] Transient expression may refer to the transient introduction of exogenous nucleic acids; for example, the exogenous nucleic acid may be introduced episomally rather than integrated into the genome. Transient expression can be achieved by incorporating a circular plasmid into cells using, for example, the calcium phosphate method, electroporation, or lipofection. Circular plasmids are less efficiently integrated into chromosomes, and the target gene is often present outside the chromosome (i.e., episomally). For this reason, it is difficult to maintain long-term expression of the target gene from a circular plasmid.

[0186] A constitutive expression system is a method in which a linear plasmid prepared by restriction enzyme treatment or the like is incorporated into cells and expressed using calcium phosphate, electroporation, lipofection, or the like, thereby stably introducing a foreign gene into cells. Linear plasmids are more efficiently inserted into chromosomes than circular plasmids, and the efficiency of maintaining the target gene on the chromosome is also higher. This makes it possible to maintain the expression of the target gene for a long period of time. Furthermore, introducing a drug resistance gene into the plasmid enables drug selection, allowing efficient selection of cells in which the target gene is maintained on the chromosome. Animal cells used in constitutive expression systems include CHO cells, NS0 cells, SP2 / 0 cells, etc., with CHO cells being preferred.

[0187] Furthermore, to achieve stable gene expression and increase the intracellular copy number of a gene, one method involves introducing a vector (e.g., pCHOI) containing a complementary DHFR gene into CHO cells lacking a nucleic acid synthesis pathway and amplifying the gene with methotrexate (MTX). To achieve transient gene expression, one method involves transforming COS cells carrying a gene expressing SV40 T antigen on their chromosomes with a vector (e.g., pcD) containing an SV40 replication origin. Replication origins derived from polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can also be used. Furthermore, to increase the gene copy number in a host cell system, the expression vector can contain a selectable marker, such as the aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, or dihydrofolate reductase (dhfr) gene.

[0188] When introducing a foreign nucleic acid into the genome of a cell, there are no particular limitations on the location of introduction on the genome, and it can be introduced at one or multiple locations, and it can be introduced at a specific target site or at a random location.

[0189] Preferably, a total of four coding regions encoding four polypeptides, two heavy chains and two light chains, are inserted at least at the same site in the genome. Insertion at least at the same site in the genome means that the four coding regions are inserted at the same site in the genome (site A), and the four coding regions may also be inserted at another site in the genome (site B). In this case, the total copy number of the four coding regions contained in the genome of the cell may be 1:1:1:1. Preferably, the four coding regions are inserted at a 1:1:1:1 ratio at each insertion site in the genome. That is, when inserted at sites A and B in the genome, they are also inserted at site A at a 1:1:1:1 ratio and at site B at a 1:1:1:1 ratio. In one embodiment, the four coding regions are inserted at one, two, three, or four sites in the genome.

[0190] Although the insertion into the same site in the genome is considered necessary, because the genome is long, it is sufficient that the insertions are at the same site from a macroscopic perspective, and it is not necessary that the insertions be at the same site from a microscopic (strict) perspective. In other words, the four coding regions encoding the four polypeptides (two heavy chains and two light chains) do not necessarily need to be inserted contiguously. That is, although the four coding regions are inserted into essentially the same site in the host cell genome, the coding regions are not contiguous, and host cell genomic sequences may be present between them. When the four coding regions are inserted into the same site in the genome, the total length of the genomic sequences from one end coding region to the other end coding region in the four coding regions on the genome preferably does not exceed 100 kb, more preferably 50 kb or less, 40 kb or less, 30 kb or less, 20 kb or less, 10 kb or less, 8 kb or less, 7 kb or less, 6 kb or less, 5 kb or less, 4 kb or less, 3 kb or less, 2 kb or less, or 1 kb or less. Preferably, no fragments of the host cell genome are present between the coding regions. If the four coding regions are inserted into other sites in the genome, a longer genomic sequence may exist between the sites. For example, between the sites A and B, a genomic sequence of more than 100 kb may exist, or the region may be inserted into a genome on a different chromosome.

[0191] In one embodiment, the foreign nucleic acid is inserted into a genomic hotspot. For introduction into a genomic hotspot, see, for example, WO2000 / 017337A1, WO2008 / 151219A1, WO2013 / 190032A1, WO2016 / 064999A1, WO2020 / 204055A1, etc.

[0192] When a total of four coding regions encoding four polypeptides, two heavy chains and two light chains, are inserted, there is no limitation on the sequence and the sequence can be determined arbitrarily.

[0193] The genome may contain one copy of each of the four coding regions encoding four polypeptides, two different heavy chains and two different light chains, or multiple copies, i.e., two or more copies. Even when multiple copies are present, the order of the coding regions is not particularly limited. For example, a preferred embodiment may include one copy of each of the four coding regions (total of 4 ORFs), two copies of each (total of 8 ORFs), or more at a certain location in the genome, such as four copies of each (total of 16 ORFs). One or more copies of the four coding regions may also be present at another location or locations in the genome.

[0194] Furthermore, without being limited thereto, for each location in the genome where the four coding regions are inserted, at least one set of sense strands of the four coding regions (i.e., four sense strands) can be included on the same strand of the double-stranded DNA of the genome. Note that this applies to each location in the genome; if the coding regions are inserted at other locations in the genome, they may be present on different strands (complementary strands) rather than the same strand, even on the same chromosome, or they may be present on different chromosomes. Furthermore, when at least one set of sense strands of the four coding regions is included on one strand of the double-stranded DNA of the genome, at least one set of sense strands of the four coding regions can also be included on the other strand of the double-stranded DNA of the genome.

[0195] The four coding regions encoding the four polypeptides (two heavy chains and two light chains) may be transcribed as separate mRNA molecules, or some or all of them may be transcribed as a single mRNA molecule. For example, by using an internal ribosome entry site (IRES), multiple coding sequences can be translated from a single RNA. For example, by transcribing an RNA in which the coding regions of a first heavy chain (H1) and its corresponding light chain (L1) are arranged in an "H1-IRES-L1" configuration, not only H1 but also L2 can be translated via the IRES. In the present invention, the four coding regions encoding the four polypeptides (two heavy chains and two light chains) may be contained in a 1:1:1:1 ratio in this manner.

[0196] Preferably, the four coding regions encoding the four polypeptides (two heavy chains and two light chains) are present on the genome as independent transcription units, and are transcribed as separate mRNA molecules. To achieve this, for example, each coding region can be expressed from its own promoter. That is, each coding region can be constructed as a separate expression unit. Here, an expression unit refers to a nucleic acid configuration for expressing one transcription unit, and typically includes a promoter, a coding sequence, and a transcription termination sequence. The transcription termination sequence may be a poly(A) addition signal. The expression unit may also include an enhancer, etc., as appropriate.

[0197] In this case, the promoter used for each transcription unit may be selected appropriately, but the same promoter may be used for the four coding regions encoding two heavy chains and two light chains. However, if multiple sets of four coding regions are included, the same promoter may be used within each set, and different promoters may be used in different sets. For example, in the first set of four coding regions encoding two heavy chains and two light chains, promoter A may be used for all four, and in the second set, promoter B may be used for all four. Of course, the same promoter may be used for both.

[0198] The promoter can be selected appropriately depending on the host cell, and for example, in the case of eukaryotes, the pol II promoter can be used. Preferred promoters for eukaryotes include, but are not limited to, the CMV promoter, CAG promoter, EF1a promoter, RSV promoter, and SV40 promoter. More specifically, promoters containing the actin promoter can be used, for example, a promoter containing the β-actin promoter, more specifically, a promoter containing the chicken β-actin promoter.

[0199] Furthermore, promoters can be appropriately combined with enhancers. Examples of enhancers include the CMV-IE enhancer, and a combination of the chicken β-actin promoter and the CMV-IE enhancer is preferred. A specific promoter is the CAG promoter (Niwa et al., Gene. (1991) 108, 193). The CAG promoter can be used in combination with the polyA signal of the rabbit β-globin gene, but this is not necessarily limited thereto. For example, polyA addition signals such as the mouse β-globin polyA signal, rabbit β-globin polyA signal, bovine growth hormone (bgh) polyA signal, or SV40 polyA signal can be used.

[0200] The present invention also provides vectors useful for producing the cells of the present invention. As used herein, the term "vector" refers to a carrier carrying a nucleic acid. For example, a vector is a nucleic acid molecule capable of carrying a nucleic acid therein or a carrier containing such a nucleic acid molecule. A vector may be a self-replicating vector or a vector that integrates into the genome of a host cell into which it is introduced. Some vectors are capable of expressing an operably linked nucleic acid. Such vectors are also referred to as "expression vectors." The form of the vector is not particularly limited, and may be a DNA vector, a viral vector, a liposomal vector, or the like. It can be introduced into host cells via viral infectivity or by well-known nucleic acid transfer methods such as electroporation. Furthermore, vector introduction is not limited to in vitro methods. For example, a vector can also be directly introduced into a subject using in vivo methods.

[0201] The vector used to produce the cells can contain multiple coding regions. For example, several of the four coding sequences encoding four polypeptides, two heavy chains and two light chains, can be carried in one vector.

[0202] In this case, there is no limit to which coding regions are included in one vector and can be determined arbitrarily. For example, if an antibody contains two heavy chains and two light chains, with a first heavy chain (H1) and a first light chain (L1) pairing to form a binding domain for a first antigen and a second heavy chain (H2) and a second light chain (L2) pairing to form a binding domain for a second antigen, one vector may contain the coding regions for the H1 and L1 polypeptides, while another vector may contain the coding regions for the H2 and L2 polypeptides. By introducing these two vectors into cells, nucleic acids encoding the coding regions for four polypeptides, H1, L1, H2, and L2, can be introduced into the cells. By introducing each vector at a 1:1 ratio, it is easy to select a vector with a 1:1:1:1 copy ratio of the H1:L1:H2:L2 coding sequences introduced into the cells.

[0203] As with the arrangement on the genome, there are no particular restrictions on the order on the vector of the coding sequences carried on the vector, and the four coding regions H1, L1, H2, and L2 may be contained in any order.

[0204] Furthermore, each coding region may be contained in a vector in one copy or multiple copies, i.e., two or more copies. When multiple copies are contained, there is no particular limitation on the order in which they are contained. For example, when two copies of the H1 and L1 coding regions are contained, the total of four coding regions may be contained in any order, and H2 and L2 may or may not be contained between them.

[0205] The same applies to H2 and L2, which may contain one copy each or multiple copies, i.e., two or more copies. When multiple copies are contained, there are no particular restrictions on the order in which they are contained. For example, when two copies of the H2 and L2 coding regions are contained, the total of four coding regions may be contained in any order, and H1 and L1 may or may not be contained between them.

[0206] Alternatively, one vector may contain the coding regions for H1 and H2, while another vector may contain the coding regions for L1 and L2. By introducing these two vectors into cells, nucleic acids encoding the coding regions for the four polypeptides H1, L1, H2, and L2 can be introduced into the cells. By introducing each vector at a 1:1 ratio, it is easy to select a vector in which the copy number ratio of the H1:L1:H2:L2 coding sequences introduced into the cells is 1:1:1:1.

[0207] When a single vector contains four coding regions encoding four polypeptides, two heavy chains and two light chains, the order of the coding sequences to be contained in the vector is not particularly limited and can be determined arbitrarily, as with the genomic arrangement described above. For example, when an antibody contains two heavy chains and two light chains, with a first heavy chain (H1) paired with a first light chain (L1) and a second heavy chain (H2) paired with a second light chain (L2), if multiple copies of each are contained, these coding regions may be arranged in any order. For example, when two copies of the coding regions for the H1 and L1 polypeptides are contained, there is no restriction on the order, and H2 or L2 may or may not be present between the coding regions. The same applies to H2 and L2; they may be present in one copy or multiple copies, i.e., two or more copies. When multiple copies are contained, there is no restriction on the order, and H1 or L1 may or may not be present between the coding regions.

[0208] The relationship between the coding regions of the H1 and H2 polypeptides is also similar, and there are no particular limitations on the order of the coding sequences. Each coding region may be contained in a single copy in the vector, or multiple copies, i.e., two or more copies. When multiple copies are contained, there are no particular limitations on the order. Furthermore, L1 and L2 may or may not be present between these coding regions.

[0209] The same applies to L1 and L2; they may each be contained in one copy or in multiple copies, i.e., two or more copies. When multiple copies are contained, there are no particular restrictions on the order. Furthermore, H1 and H2 may or may not be contained between these coding regions.

[0210] Furthermore, in the present invention, a vector containing coding regions for four polypeptides, i.e., two heavy chains (H1 and H2) and two light chains (L1 and L2), of an antibody, has been found to be extremely useful. In one preferred embodiment of the present invention, a single vector may contain the coding regions for the four polypeptides H1, L1, H2, and L2.

[0211] Specifically, the present invention provides a recombinant vector containing, in a copy ratio of 1:1:1:1, four polypeptide coding regions encoding two antibody heavy chains or antigen-binding fragments thereof and two light chains or antigen-binding fragments thereof corresponding to each heavy chain. The present invention also provides a recombinant vector containing, in equal copy numbers, four polypeptide coding regions encoding two antibody heavy chains or antigen-binding fragments thereof and two light chains or antigen-binding fragments thereof corresponding to each heavy chain. When the vector encodes antigen-binding fragments, in a preferred embodiment, the two antigen-binding fragments derived from the two heavy chains encoded by the vector are different from each other, and the two antigen-binding fragments derived from the two light chains encoded by the vector are also different from each other. By containing, in equal copy numbers, the coding regions for the four polypeptides H1, L1, H2, and L2 in a single vector, i.e., in a 1:1:1:1 ratio, it is possible to easily produce cells transfected with a 1:1:1:1 copy ratio of the H1:L1:H2:L2 coding regions.

[0212] For example, the four coding regions encoding the four polypeptides, two heavy chains (H1, H2) and two light chains (L1, L2), may each be contained in a single copy or in multiple copies, i.e., two or more copies, in a vector. Even when multiple copies are contained, the order of the coding regions is not particularly limited. For example, vectors containing one copy, two copies, three copies, four copies, or five copies of each of the four coding regions, H1, L1, H2, and L2, are preferred vectors of the present invention. A preferred example is a vector containing one copy of each of the coding regions for the four polypeptides encoding the two heavy chains and two light chains (i.e., a total of four coding regions). Another preferred example is a vector containing two copies of each of the coding regions for the four polypeptides encoding the two heavy chains and two light chains (i.e., a total of eight coding regions).

[0213] In a vector containing coding regions for four polypeptides, i.e., two heavy chains (H1 and H2) and two light chains (L1 and L2), the order of the coding sequences is not particularly limited and can be arranged in any order. As described above for the arrangement on the genome, the four coding regions can be arranged in any desired order.

[0214] Furthermore, in a vector into which the four coding regions have been inserted, the sense strands of the four coding regions (or the antisense strands, if they are encoded as antisense, as in a minus-strand viral vector) for each set of four coding regions can be contained on the same strand of the nucleic acid of the vector, although this is only for each set of four coding regions. If another set (a second set) of four coding regions is inserted at another location in the vector, the sense strand of the coding region of the second set may be a different strand (complementary strand) from that of the first set, or may be the same strand, even if contained on the same vector as the first set.

[0215] The coding regions for four polypeptides encoding two heavy chains and two light chains may be transcribed from a vector as separate mRNA molecules, or some or all of them may be transcribed as a single mRNA molecule. For example, by using an internal ribosome entry site (IRES), multiple coding sequences can be translated from a single RNA. For example, by transcribing an RNA in which the coding regions for a first heavy chain (H1) and its corresponding light chain (L1) are arranged in an "H1-IRES-L1" configuration, not only H1 but also L2 can be translated via the IRES. In the present invention, the coding regions for four polypeptides encoding two heavy chains and two light chains may be contained in a vector in a 1:1:1:1 ratio in this manner.

[0216] Preferably, the four coding regions encoding the four polypeptides (two heavy chains and two light chains) are present on a vector as independent transcription units, and each is transcribed as a different mRNA molecule. To achieve this, for example, each coding region can be expressed from its own promoter. That is, each coding region can be constructed as a separate expression unit. As described above, an expression unit is a nucleic acid construct for expressing a single transcription unit, and typically includes a promoter, a coding sequence, and a transcription termination sequence (poly(A) addition signal).

[0217] In this case, the promoter used for each transcription unit may be selected appropriately, but the same promoter may be used for the four coding regions encoding the four polypeptides, i.e., two heavy chains and two light chains. However, if multiple sets of four coding regions are included, the same promoter may be used within each set (i.e., within the four coding sequences), and different promoters may be used in different sets. For example, in the first set of four coding regions encoding two heavy chains and two light chains, promoter A may be used for all four, and in the second set, promoter B may be used for all four. Of course, the same promoter may be used for both.

[0218] In one preferred embodiment of the vector, two copies of the coding regions for four polypeptides encoding two heavy chains and two light chains are contained on a single vector (i.e., a total of eight coding regions encoding antibody chains are contained). The vector may be, for example, a plasmid, and the sense strands of all coding regions encoding antibody chains may be the same strand of the plasmid. A promoter may be linked upstream of each coding region encoding an antibody chain. A transcription termination signal (e.g., a poly(A) addition signal) may be linked downstream of each coding region encoding an antibody chain. The promoter-coding region-transcription termination signal constitutes a single transcription unit (also an expression unit). The above-mentioned plasmid contains eight transcription units (also expression units) encoding antibody chains. The plasmid may further contain expression units for one or more drug resistance genes or marker genes. In another embodiment, the vector of the present invention contains one copy of the coding regions for four polypeptides encoding two heavy chains and two light chains. The vector may be, for example, a plasmid, and the sense strands of all coding regions encoding antibody chains may be the same strand of the plasmid. Furthermore, a promoter may be ligated upstream of each coding region encoding the antibody chain. Such a vector contains four transcription units (which are also expression units) encoding the antibody chains. The plasmid may further contain expression units for one or more drug resistance genes or marker genes.

[0219] The promoter can be selected appropriately depending on the host cell into which the vector is introduced. As described above, for example, in the case of eukaryotes, the promoter can be the Pol II promoter. Preferred promoters for eukaryotes include, but are not limited to, the CMV promoter, CAG promoter, EF1a promoter, RSV promoter, and SV40 promoter. More specifically, promoters containing the actin promoter can be used, such as a promoter containing the β-actin promoter, and more specifically, a promoter containing the chicken β-actin promoter can be preferably used.

[0220] Furthermore, promoters can be appropriately combined with enhancers. Examples of enhancers include the CMV-IE enhancer, and a combination of the chicken β-actin promoter and the CMV-IE enhancer is preferred. A specific promoter is the CAG promoter (Niwa et al., Gene. (1991) 108, 193). The CAG promoter can be used in combination with the polyA signal of the rabbit β-globin gene, but this is not necessarily limited thereto. For example, polyA addition signals such as the mouse β-globin polyA signal, rabbit β-globin polyA signal, bovine growth hormone (bgh) polyA signal, or SV40 polyA signal can be used.

[0221] The present invention also relates to host cells into which exogenous nucleic acid encoding the vector of the present invention or the antibody of the present invention has been introduced. Here, "host cell" refers to a "cell" (including the progeny of such a cell) into which exogenous nucleic acid has been introduced. Host cells may also be referred to as "transformants" or "transformed cells," and include the original transformed cell and its progeny, regardless of the number of passages. The progeny contain copies of the exogenous nucleic acid introduced into the cell. These copies are also referred to as exogenous nucleic acid. Host cells may be cultured cells or established cell lines (host cell lines). Furthermore, the progeny need not be completely identical to the parent cell in terms of nucleic acid content and may contain mutations. Naturally, mutant progeny that have the same function or biological activity as that used to screen or select the original transformed cell are also included in the term "transformed cells."

[0222] There are no particular limitations on the host cells into which the vectors of the present invention are introduced, and for example, Escherichia coli and various animal cells can be used. The host cells of the present invention can be used, for example, as a production system for producing or expressing the antibodies of the present invention or fragments thereof. Production systems for producing polypeptides include in vitro and in vivo production systems. In vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.

[0223] There are no particular limitations on the type or copy number of vectors introduced into cells. One type of vector may be introduced, or two or more types of vectors may be introduced into a single cell. Furthermore, only one copy of a vector may be introduced into a cell, or two, three, four, five, or more copies may be introduced.

[0224] As already mentioned, the vector may be transiently introduced or may exist episomally. Preferably, the vector is stably introduced. To this end, the vector can be introduced into the genome of the host cell. In this case, the vector may be introduced at one site on the genome, or at two or more sites. Furthermore, at each site, one copy of the vector may be introduced, or two, three, four, five, or more copies may be introduced.

[0225] When multiple copies of a vector are inserted into a single location in the genome, these copies do not need to be inserted contiguously. That is, multiple copies of the vector are inserted into substantially the same site in the genome, but the vectors are not linked, and a genomic sequence or a desired intervening sequence may be present between them. When multiple vectors are inserted into a single location in the genome, the total length of the genomic sequence and / or desired intervening sequence from one end vector to the other end vector preferably does not exceed 100 kb, and more preferably is 50 kb or less, 40 kb or less, 30 kb or less, 20 kb or less, 10 kb or less, 8 kb or less, 7 kb or less, 6 kb or less, 5 kb or less, 4 kb or less, 3 kb or less, 2 kb or less, or 1 kb or less. When multiple copies of a vector are inserted into a single location, preferably, no genomic fragments are present between the multiple copies of the vector. Note that this applies per site; in addition to the first site in the genome, the vector may also be introduced into another site (second site), and the second site may be far away from the first site (e.g., more than 100 kb away) or may be on a different chromosome.

[0226] For example, one preferred embodiment includes a cell having two copies of a vector at a certain location in its genome. A particularly preferred embodiment includes a cell having two copies of the vector, each of which contains two copies of the coding regions for four polypeptides encoding the two heavy chains and two light chains of an antibody (comprising a total of eight antibody chain coding regions), and two copies of this vector introduced into at least one location in its genome (comprising a total of 16 antibody chain coding regions). Another embodiment includes a cell having one copy of the vector, each of which contains one copy of the coding regions for four polypeptides encoding the two heavy chains and two light chains of an antibody (comprising a total of four antibody chain coding regions), and two copies of this vector introduced into at least one location in its genome (comprising a total of eight antibody chain coding regions).

[0227] In one embodiment, the vector is inserted into a hotspot in the genome. Hotspot refers to a region in the genome that shows a recombination rate higher than the normal expected value. For the introduction of foreign nucleic acid into a hotspot in the genome, refer to, for example, WO2000 / 017337A1, WO2008 / 151219A1, WO2013 / 190032A1, WO2016 / 064999A1, WO2020 / 204055A1, etc.

[0228] The vector can optionally contain a site-specific recombination sequence such as loxP or FRT. This allows for accurate introduction into the target site in the genome. For example, if the vector contains multiple antibody chain coding sequences, site-specific recombination sequences such as loxP or FRT can be placed at both ends of the antibody chain coding region group, i.e., before the first coding sequence and after the last coding sequence. This allows multiple coding sequences in the vector to be inserted into the genome together. If the vector is circular, the ends of the antibody chain coding region group can be determined by assuming that the vector has been linearized by cleavage at the region containing the longest continuous sequence other than the antibody chain coding sequence (the original sequence of the vector before insertion of the antibody chain coding sequence).

[0229] The cells used in the present invention for expression of antibodies or fragments thereof are not particularly limited, and may be any cells, including animal cells, plant cells, eukaryotic cells such as yeast, and prokaryotic cells such as Escherichia coli and Bacillus subtilis. Animal cells such as CHO cells (J. Exp. Med. (1995) 108, 945), COS cells, 3T3 cells, myeloma cells, BHK (baby hamster kidney) cells, HeLa cells, Vero cells, and lymphoid cells (e.g., Y0, NS0, and Sp2 / 0 cells), with vertebrate cells, such as mammalian cells, being preferred. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68). (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR －Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Rodent cells, such as Chinese hamsters, are preferred, particularly Chinese hamster ovary (CHO) cells. These cells may be established or non-established, e.g., primary culture cells. They may also be in vitro or ex vivo. For production of the desired antibody, preferred cells are those suitable for introducing a desired gene, such as CHO dhfr- cells. For example, it is possible to culture COS cells or CHO cells into which a gene encoding a desired antibody has been inserted by genetic engineering.

[0230] CHO cells that are particularly suitable for use are dhfr-CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220), which are CHO cells lacking the DHFR gene, and CHO K-1 (Proc. Natl. Acad. Sci. USA (1968) 60, 1275). CHO cells are particularly preferred for large-scale expression in animal cells. Introduction of vectors into host cells can be carried out by, for example, the calcium phosphate method, the DEAE-dextran method, a method using cationic liposome DOTAP (Boehringer Mannheim), electroporation, lipofection, and the like.

[0231] For example, when Escherichia coli is used as a host, vectors that can be used in the methods of the present invention preferably have an origin of replication (ori) for amplification in E. coli (e.g., JM109, DH5α, HB101, XL1Blue) and a selection gene for transformed E. coli (e.g., a drug resistance gene that can be detected by the treatment with a drug (ampicillin, tetracycline, kanamycin, chloramphenicol)) in order to amplify and prepare the vector in large quantities. Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script. In addition to the above vectors, other vectors that can be used for the purpose of subcloning and excision of cDNA include, for example, pGEM-T, pDIRECT, and pT7. When using vectors for the purpose of producing the antibodies or fragments thereof of the present invention, expression vectors are particularly useful. For example, when expression in E. coli is desired, the expression vector preferably has the above-mentioned characteristics that allow the vector to be amplified in E. coli. In addition, when the host is E. coli such as JM109, DH5α, HB101, or XL1-Blue, it preferably has a promoter that allows efficient expression in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), the araB promoter (Better et al., Science (1988) 240, 1041-1043), or the T7 promoter. In addition to the above vectors, other such vectors include pGEX-5X-1 (Pharmacia), the "QIAexpress system" (Qiagen), pEGFP, and pET (in this case, the host is preferably BL21, which expresses T7 RNA polymerase).

[0232] The vector may also contain a signal sequence for polypeptide secretion. When producing a polypeptide in the periplasm of E. coli, the signal sequence for polypeptide secretion may be the pelB signal sequence (Lei, SP et al. J. Bacteriol. (1987) 169, 4379). Introduction of the vector into host cells can be achieved, for example, by the calcium chloride method or electroporation.

[0233] An example of a prokaryotic cell other than E. coli is Bacillus subtilis.

[0234] For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste and can be further purified.

[0235] In addition to prokaryotic cells, various other hosts can be used. For example, vectors that can be used in the methods of the present invention include mammalian expression vectors (e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322), pEF, pCDM8, and INPEP4 (Biogen-IDEC)), insect cell-derived expression vectors (e.g., the "Bac-to-BAC baculovairus expression system" (GIBCO BRL), pBacPAK8), plant-derived expression vectors (e.g., pMH1, pMH2), viral vectors, particularly animal virus-derived expression vectors (e.g., pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (e.g., pZIpneo), yeast-derived expression vectors (e.g., the "Pichia Expression Kit" (Invitrogen), pNV11, SP-Q01), and Bacillus subtilis-derived expression vectors (e.g., pPL608, pKTH50).

[0236] For expression in animal cells such as CHO cells, COS cells, and NIH3T3 cells, promoters necessary for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), the CMV promoter (Niwa et al., Gene. (1991) 108, 193), β-actin promoters such as chicken β-actin promoter, and promoters in which the cytomegalovirus enhancer (CMV-IE) has been added to the β-actin promoter (for example, the CAG promoter (Niwa, H et al., Gene 108: 193-200, Preferably, the vector contains a gene for selecting cells for transformation (e.g., a drug resistance gene that can be detected by drugs (neomycin, G418, etc.)). Examples of vectors with such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13. It is known that mRNA containing polyA is stable within cells, and it is preferable for the vector to contain a polyA signal required for adding polyA to a gene, such as a mouse β-globin polyA signal, a rabbit β-globin polyA signal, a bovine growth hormone polyA signal, or an SV40 polyA signal. Particularly preferred combinations include, but are not limited to, the combination of a CAG promoter and a rabbit β-globin polyA signal, and the combination of a CAG promoter and a bovine growth hormone polyA signal.

[0237] Known host cells include amphibian cells, such as Xenopus oocytes (Valle, et al., Nature (1981) 291, 358-340), and insect cells, such as Sf9, Sf21, and Tn5.

[0238] For example, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns, are suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0239] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly transformation of Spodoptera frugiperda cells.

[0240] For the use of plant cell cultures as hosts, see, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0241] As plant cells, for example, cells derived from Nicotiana tabacum are known as a polypeptide production system, and these can be cultured as calli. As fungal cells, yeasts such as Saccharomyces, e.g., Saccharomyces cerevisiae, and filamentous fungi such as Aspergillus, e.g., Aspergillus niger, are known.

[0242] These cells are transformed with a gene of interest and the transformed cells are cultured in vitro to obtain a polypeptide encoded by the gene of interest. Culture can be performed according to known methods. For example, DMEM, MEM, RPMI1640, or IMDM can be used as a culture medium for animal cells. Serum supplements such as fetal calf serum (FCS) can be used in combination, or serum-free culture can be performed. The pH during culture is preferably about 6 to 8. Culture is typically performed at about 30 to 40°C for about 15 to 200 hours, with medium replacement, aeration, and agitation as necessary.

[0243] For cell culture, a medium used for conventional cell (preferably animal cell) culture can be used, which usually contains amino acids, vitamins, lipid factors, an energy source, an osmotic pressure regulator, an iron source, and a pH buffer. The contents of these components are typically within the ranges of 0.05-1500 mg / L for amino acids, 0.001-10 mg / L for vitamins, 0-200 mg / L for lipid factors, 1-20 g / L for energy sources, 0.1-10,000 mg / L for osmotic pressure regulators, 0.1-500 mg / L for iron sources, 1-10,000 mg / L for pH buffers, 0.00001-200 mg / L for trace metal elements, 0-5,000 mg / L for surfactants, 0.05-10,000 μg / L for growth cofactors, and 0.001-50 mg / L for nucleosides, but are not limited to these and can be determined appropriately depending on the type of cells to be cultured, the type of antibody or fragment thereof to be produced, and the like.

[0244] In addition to the above components, for example, trace metal elements, surfactants, growth cofactors, nucleosides, etc. may be added.

[0245] Specific examples include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and preferably L-alanine, L-arginine, and L-aspartic acid. Amino acids such as paragine, L-aspartic acid, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; i-inositol, biotin, folic acid, lipoic acid, nicotinamide, nicotinic acid, p-aminobenzoic acid, calcium pantothenate, and pyridoxine hydrochloride. lipid factors such as choline chloride, choline tartrate, linoleic acid, oleic acid, cholesterol, etc., preferably choline chloride; energy sources such as glucose, galactose, mannose, fructose, etc., preferably glucose; osmotic regulators such as sodium chloride, potassium chloride, potassium nitrate, etc., preferably sodium chloride; iron sources such as ferric EDTA, ferric citrate, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, ferric nitrate, etc., preferably ferric chloride, ferric EDTA, ferric citrate; and pH buffers such as sodium bicarbonate, calcium chloride, sodium dihydrogen phosphate, HEPES, MOPS, etc., preferably sodium bicarbonate.

[0246] In addition to the above components, trace metal elements such as copper sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, nickel chloride, tin chloride, magnesium chloride, and sodium silicate, preferably copper sulfate, zinc sulfate, and magnesium sulfate; surfactants such as Tween® 80 and Pluronic® F68; growth cofactors such as recombinant insulin, recombinant IGF-1, recombinant EGF, recombinant FGF, recombinant PDGF, recombinant TGF-α, ethanolamine hydrochloride, sodium selenite, retinoic acid, and putrescine hydrochloride, preferably sodium selenite, ethanolamine hydrochloride, recombinant IGF-1, and putrescine hydrochloride; and nucleosides such as deoxyadenosine, deoxycytidine, deoxyguanosine, adenosine, cytidine, guanosine, and uridine. Suitable examples of the above medium may also contain antibiotics such as streptomycin, penicillin G potassium, and gentamicin, and pH indicators such as phenol red.

[0247] The pH of the medium varies depending on the cells to be cultured, but generally it is pH 6.8 to 7.6, and in most cases pH 7.0 to 7.4.

[0248] The medium may be a commercially available medium for animal cell culture, such as D-MEM (Dulbecco's Modified Eagle Medium), D-MEM / F-12 1:1 Mixture (Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12), RPMI1640, CHO-S-SFM II (Invitrogen), CHO-SF (Sigma-Aldrich), EX-CELL 301 (JRH biosciences), CD-CHO (Invitrogen), IS CHO-V (Irvine Scientific), or PF-ACF-CHO (Sigma-Aldrich).

[0249] The medium may also be a serum-free medium.

[0250] When the cells are CHO cells, they can be cultured using methods known to those skilled in the art. For example, they can be cultured in an atmosphere with a CO concentration in the gas phase of 0 to 40%, preferably 2 to 10%, at 30 to 39°C, preferably about 37°C.

[0251] The culture period for cells suitable for producing the desired recombinant antibody or fragment thereof is usually 1 day to 3 months, preferably 1 day to 2 months, and more preferably 1 day to 1 month.

[0252] In addition, various culture apparatuses for animal cell culture may be used, such as a fermenter-type tank culture apparatus, an airlift-type culture apparatus, a culture flask-type culture apparatus, a spinner flask-type culture apparatus, a microcarrier-type culture apparatus, a fluidized bed-type culture apparatus, a hollow fiber-type culture apparatus, a roller bottle-type culture apparatus, and a packed tank-type culture apparatus.

[0253] The culture may be any of batch culture, fed-batch culture, continuous culture, etc., but fed-batch culture or continuous culture is preferred, with fed-batch culture being more preferred.

[0254] On the other hand, systems for producing antibodies or fragments thereof in vivo include, for example, production systems using animals and plants. A target gene is introduced into these animals or plants, and the polypeptide is produced in the animal or plant body and then recovered. In the present invention, the "host" includes these animals and plants.

[0255] When animals are used, there are production systems using mammals and insects. Mammals that can be used include goats, pigs, sheep, mice, and cows (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When mammals are used, transgenic animals can also be used.

[0256] Methods for producing transgenic animals are known. For example, transgenic animals can be obtained by the method described in Proc. Natl. Acad. Sci. USA 77:7380-7384 (1980). Specifically, a gene of interest is introduced into totipotent cells of a mammal, and these cells are allowed to develop into individuals. From the individuals obtained, individuals in which the transgene has been incorporated into somatic cells and germ cells are selected to produce the desired transgenic animal. Examples of totipotent cells into which a gene can be introduced include fertilized eggs and early embryos, as well as cultured cells such as pluripotent ES cells.

[0257] For example, it may be prepared as a fusion gene with a gene encoding a polypeptide specifically produced in milk, such as goat β-casein. A gene fragment containing this fusion gene is then injected into a goat embryo, and the embryo is then implanted into a female goat. The target polypeptide (in the present invention, an antibody or a fragment thereof) can be obtained from the milk produced by the transgenic goat born from the embryo-receiving goat or its offspring. To increase the amount of milk containing the polypeptide produced by the transgenic goat, appropriate hormones may be administered to the transgenic goat (Ebert, KM et al., Bio / Technology (1994) 12, 699-702).

[0258] Furthermore, as an insect, for example, silkworms can be used. When silkworms are used, the silkworms are infected with a baculovirus into which a gene encoding a desired polypeptide has been inserted, and the desired polypeptide can be obtained from the body fluids of the silkworms (Susumu, M. et al., Nature (1985) 315, 592-594).

[0259] Furthermore, when using plants, tobacco can be used. In tobacco, a gene encoding a polypeptide of interest is inserted into a plant expression vector, such as pMON 530, and this vector is then introduced into a bacterium such as Agrobacterium tumefaciens. This bacterium is then used to infect tobacco, such as Nicotiana tabacum, and the desired polypeptide (in this invention, an antibody or a fragment thereof) can be obtained from the tobacco leaves (Julian K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).

[0260] The present invention also provides a method for producing an antibody or a fragment thereof, comprising producing the antibody or a fragment thereof using cells expressing the antibody or a fragment thereof of the present invention. Examples of the cells include the cells described above. By culturing the cells, the cells can be made to produce the antibody or a fragment thereof with high efficiency. The culture medium and culture conditions are as described above.

[0261] Antibodies produced by the production method of the present invention include not only monoclonal antibodies derived from animals such as humans, mice, rats, hamsters, rabbits, and monkeys, but also artificially modified recombinant antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies. The present invention is particularly useful for producing antibodies comprising two heavy chains and two light chains, preferably bispecific antibodies. The class of the antibody is not particularly limited, and the immunoglobulin class of the antibody is not particularly limited, and any class, including IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgA, IgD, IgE, and IgM, is acceptable. However, IgG and IgM are preferred for pharmaceutical use. Furthermore, the antibodies of the present invention include not only whole antibodies but also polypeptides comprising antibodies comprising two heavy chains and two light chains or fragments thereof, including antibody fragments such as F(ab')2.

[0262] The present invention also provides an expression product produced by the above-described production method of the present invention. Specifically, the expression product may be a composition containing a molecule (polypeptide) comprising an antibody or a fragment thereof. The composition contains a population of molecules (polypeptides) comprising an antibody or a fragment thereof expressed from a cell.

[0263] The antibodies produced by the methods of the present invention can also be used as modified antibodies by conjugating them with various molecules such as polyethylene glycol (PEG). Such modified antibodies can be obtained by chemically modifying the antibodies obtained. These methods have already been established in this field.

[0264] In some embodiments, the antibody of the present invention may be post-translationally modified. Examples of post-translation modifications include pyroglutamylation at the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain. It is known in the art that such post-translational modifications by pyroglutamylation at the N-terminus and deletion of lysine at the C-terminus have no effect on the activity of the antibody (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39).

[0265] It is also known that antibodies are modified after translation when expressed in cells. Examples of post-translational modifications include cleavage of the lysine at the C-terminus of the heavy chain by carboxypeptidase; modification of glutamine or glutamic acid at the N-terminus of the heavy chain and light chain to pyroglutamic acid by pyroglutamylation; glycosylation; oxidation; deamidation; and glycation. Such post-translational modifications are known to occur in various antibodies (Journal of Pharmaceutical Sciences, 2008, Vol. 97, pp. 2426-2447). The antibody of the present invention may be modified in this way.

[0266] Furthermore, the antibodies of the present invention may be conjugated with carrier polymers such as PEG or organic compounds such as anticancer drugs. Alternatively, glycosylation sequences are suitably inserted into the antibodies so that the glycosylation effects are desired.

[0267] When a linker is used to link the antibody variable regions, the linker may be any peptide linker that can be introduced by genetic engineering, a synthetic linker, or a linker disclosed in, for example, Protein Engineering, 9 (3), 299-305, 1996. However, in the present disclosure, a peptide linker is preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose.

[0268] The invention also provides immunoconjugates comprising an antibody conjugated to one or more cytotoxic agents (e.g., a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin of bacterial, fungal, plant, or animal origin, an enzymatically active toxin, or fragment thereof), or a radioactive isotope).

[0269] Alternatively, in the case of multispecific antibodies, even if the target molecule cannot be efficiently formed, it is possible to separate and purify the target molecule from the produced antibody. For example, as described above, by introducing amino acid substitutions into the variable regions of two types of H chains to impart a difference in isoelectric point, it is possible to purify two types of homoantibodies and the target heteroantibody by ion exchange chromatography (WO2007114325). A method for purifying heteroantibodies has been reported in the past, in which a heterodimerized antibody containing a mouse IgG2a H chain that binds to Protein A and a rat IgG2b H chain that does not bind to Protein A is purified using Protein A (WO98050431 and WO95033844).

[0270] Furthermore, by using an H chain in which the amino acid residues at EU numbering positions 435 and / or 436, which are the binding sites between IgG and Protein A, are substituted with amino acids such as Tyr or His that confer different Protein A affinities, or by using H chains with different Protein A affinities, the interaction between each H chain and Protein A can be altered, and then using a Protein A column, it is possible to efficiently purify only the heterodimerized antibody.

[0271] The recombinant antibody produced by the method of the present invention is not particularly limited and may be a recombinant antibody against any antigen, such as an anti-IL-6 receptor antibody, anti-IL-6 antibody, anti-glypican-3 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-GPIIb / IIIa antibody, anti-TNF antibody, anti-CD25 antibody, anti-EGFR antibody, anti-Her2 / neu antibody, anti-RSV antibody, anti-CD33 antibody, anti-CD52 antibody, anti-IgE antibody, anti-CD11a antibody, anti-VEGF antibody, anti-VLA4 antibody, etc. As already described, in one embodiment, the antigen molecule is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ionotropic receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, MHC molecules, growth factors or receptors thereof, growth factors or receptors thereof, and cell adhesion molecules. Specific examples include, but are not limited to, membrane proteins constituting tight junctions, T cell receptor (TCR) complex proteins, TNFR (tumor necrosis factor receptor) superfamily members, MHC class II (HLA class II) molecules (including HLA-DR, DQ, DP, etc.), platelet-derived growth factors (PDGFs) (including PDGF-A, PDGF-B, PDGF-C, PDGF-D, etc.) or their receptors.

[0272] Examples of cell adhesion molecules include, but are not limited to, claudin family members such as Claudin 6; examples of TCR complex-forming proteins include CD3, particularly CD3ε; examples of MHC class II (HLA class II) molecules include HLA-DQ2.5, HLA-DQ7.5, HLA-DQ5.1, HLA-DQ6.3, HLA-DQ7.3, and HLA-DQ8, particularly HLA-DQ2.5; and examples of PDGFs include PDGF-B and PDGF-D.

[0273] For example, the antibodies of the present invention can be suitably applied to multispecific antibodies that bind to a complex formed by HLA-DQ2.5 and a gluten peptide. As used herein, a multispecific antibody that binds to a complex formed by HLA-DQ2.5 and a gluten peptide refers to an antibody that binds to at least two complexes formed by HLA-DQ2.5 and a gluten peptide, i.e., complex A formed by HLA-DQ2.5 and a gluten peptide, and complex B formed by HLA-DQ2.5 and a gluten peptide. Here, the gluten peptide contained in complex A is a different peptide from the gluten peptide contained in complex A. For example, a bispecific antibody that binds to a complex formed by HLA-DQ2.5 and a gluten peptide refers to an antibody that binds to two complexes, complex A formed by HLA-DQ2.5 and a gluten peptide and complex B formed by HLA-DQ2.5 and a gluten peptide different from the gluten peptide.

[0274] Specifically, the antibody specifically recognizes a complex of HLA-DQ2.5 and a gluten peptide, and preferably has substantially no binding activity to either or both of HLA-DQ2.5-positive PBMC B cells and HLA-DQ2.5-expressing Ba / F3 cells. The gluten peptide is preferably an immunodominant peptide associated with celiac disease, for example, a 33-mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4a gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, or a β-gliadin peptide. The gluten peptide is selected from the group consisting of ω2 gliadin peptide, BC hordein peptide, γ1 gliadin peptide, γ2 gliadin peptide, γ4a gliadin peptide, and γ4d gliadin peptide (WO2022 / 059766; JP2022-051553).

[0275] Furthermore, the multispecific antibodies used in the present invention that bind to a complex formed by HLA-DQ2.5 and gluten peptides preferably have substantially no binding activity to HLA-DQ2.5 in a complex with a peptide unrelated to the gluten peptide, such as at least one peptide selected from the group consisting of CLIP peptides, Hepatitis B virus 1 peptides, Salmonella peptides, Mycobacterium bovis peptides, and thyroperoxidase peptides.

[0276] Furthermore, the multispecific antibodies used in the present invention that bind to the complex formed by HLA-DQ2.5 and gluten peptides are preferably humanized, and their amino acid sequences are preferably modified so that their binding activity to the complex formed by HLA-DQ2.5 and gluten peptides is enhanced.

[0277] Specifically, for example, one, two, three, or all of the sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in the following (a) to (d) in the antibody heavy chain and light chain may be modified to amino acid residues that are electrostatically repulsive to each other: (a) the amino acid residue in the heavy chain constant region (CH1) at position 175 according to EU numbering and the amino acid residue in the light chain constant region (CL) at position 131 according to Kabat numbering, (b) the amino acid residue in CH1 at position 175 according to EU numbering and the amino acid residue in CL at position 160 according to Kabat numbering, (c) the amino acid residue in CH1 at position 175 according to EU numbering and the amino acid residues in CL at positions 131 and 160 according to Kabat numbering, (d) the amino acid residues in CH1 at positions 147 and 175 according to EU numbering and the amino acid residues in CL at positions 131 and 160 according to Kabat numbering.

[0278] Furthermore, it is also preferred that two or more amino acid residues forming the interface between the heavy-chain variable region and the light-chain variable region are mutually electrostatically repulsive amino acid residues. The mutually electrostatically repulsive amino acid residues may be one or two sets of amino acid residues selected from the group consisting of the following sets of amino acid residues (a) and (b): (a) the amino acid residue in the heavy-chain variable region at position 39 according to the Kabat numbering and the amino acid residue in the light-chain variable region at position 38 according to the Kabat numbering, and (b) the amino acid residue in the heavy-chain variable region at position 45 according to the Kabat numbering and the amino acid residue in the light-chain variable region at position 44 according to the Kabat numbering.

[0279] The mutually electrostatically repulsive amino acid residues may be selected from amino acid residues included in either the following sets (X) or (Y), as described herein: (X) glutamic acid (E), aspartic acid (D), (Y) lysine (K), arginine (R), histidine (H).

[0280] The antibody may also have an Fc domain that exhibits reduced binding affinity to human Fcγ receptors compared to a native human IgG1 Fc domain. For example, the Fc domain may contain Arg at positions 235 and 236 (EU numbering).

[0281] Alternatively, the Fc domain may be composed of a first Fc region subunit and a second Fc region subunit capable of stable association. For example, the Fc domain may include the following (e1) or (e2) (amino acid positions are according to EU numbering): (e1) a first Fc region subunit including Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407, and a second Fc region subunit including Cys at position 354 and Trp at position 366; (e2) a first Fc region subunit including Glu at position 439, and a second Fc region subunit including Lys at position 356.

[0282] The Fc domain may also exhibit stronger FcRn-binding affinity to human FcRn than native human IgG1 Fc domains. For example, the first and / or second Fc region subunit may contain Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (amino acid positions are according to EU numbering).

[0283] Furthermore, it is preferable that the antibody has substantially no binding activity to the gluten peptide itself.

[0284] More specifically, examples of multispecific antibodies that bind to the complex formed by HLA-DQ2.5 and gluten peptides include those that contain one or more of the following amino acid residues (i) to (xii): (i) a glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region; (ii) a glutamic acid at position 147 (EU numbering) in the heavy chain constant region; (iii) a glutamic acid or lysine at position 131 (Kabat numbering) in the light chain constant region; (iv) a glutamic acid or lysine at position 160 (Kabat numbering) in the light chain constant region; (v) an arginine at position 235 (EU numbering) in the heavy chain constant region; (vi) an arginine at position 236 (EU numbering) in the heavy chain constant region; (vii) a lysine at position 356 (EU numbering) in the heavy chain constant region; (viii) (ix) alanine at position 434 (EU numbering) in the heavy chain constant region; (x) arginine at position 438 (EU numbering) in the heavy chain constant region; (xi) glutamic acid at position 439 (EU numbering) in the heavy chain constant region; (xii) glutamic acid at position 440 (EU numbering) in the heavy chain constant region.

[0285] The antibody is, for example, a bispecific antibody comprising: a first heavy chain comprising a lysine at position 175 (EU numbering), an arginine at position 235 (EU numbering), an arginine at position 236 (EU numbering), a leucine at position 428 (EU numbering), an alanine at position 434 (EU numbering), an arginine at position 438 (EU numbering), a glutamic acid at position 439 (EU numbering), and a glutamic acid at position 440 (EU numbering); a first light chain comprising a glutamic acid at position 131 (Kabat numbering) and a glutamic acid at position 160 (Kabat numbering); a second heavy chain comprising a glutamic acid at position 147 (EU numbering), a glutamic acid at position 175 (EU numbering), an arginine at position 235 (EU numbering), an arginine at position 236 (EU numbering), a lysine at position 356 (EU numbering), a leucine at position 428 (EU numbering), an alanine at position 434 (EU numbering), an arginine at position 438 (EU numbering), and a glutamic acid at position 440 (EU numbering); and a second light chain comprising a lysine at position 131 (Kabat numbering) and a lysine at position 160 (Kabat numbering).

[0286] The antibody may also have, for example, a first heavy chain that further comprises a glutamic acid at position 419 (EU numbering), a proline at position 445 (EU numbering), and amino acid deletions at positions 446 and 447 (EU numbering); and a second heavy chain that further comprises a lysine at position 196 (EU numbering), a proline at position 445 (EU numbering), and amino acid deletions at positions 446 and 447 (EU numbering).

[0287] wherein the first heavy chain further comprises a glycine at position 16 (Kabat numbering), an alanine at position 32 (Kabat numbering), a lysine at position 61 (Kabat numbering), a valine at position 35a (Kabat numbering), an alanine at position 50 (Kabat numbering), a glutamic acid at position 64 (Kabat numbering), a threonine at position 73 (Kabat numbering), a glutamic acid at position 95 (Kabat numbering), and a valine at position 102 (Kabat numbering); the first light chain further comprises a glutamic acid at position 28 (Kabat numbering), a tyrosine at position 55 (Kabat numbering), a glutamic acid or tyrosine at position 56 (Kabat numbering), a glutamic acid at position 92 (Kabat numbering), a valine at position 94 (Kabat numbering), and an alanine at position 95a (Kabat numbering); the second heavy chain contains a glutamic acid at position 28 (Kabat numbering), an alanine or glutamic acid at position 30 (Kabat numbering), a glutamic acid at position 31 (Kabat numbering), a tryptophan at position 32 (Kabat numbering), a phenylalanine at position 34 (Kabat numbering), a methionine at position 35 (Kabat numbering), a serine at position 35a (Kabat numbering), a serine at position 50 (Kabat numbering), a glutamic acid or and the second light chain further comprises threonine at position 25 (Kabat numbering), lysine at position 54 (Kabat numbering), glutamic acid at position 56 (Kabat numbering), leucine at position 67 (Kabat numbering), glutamine at position 79 (Kabat numbering), and lysine at position 94 (Kabat numbering).

[0288] More specifically, examples of the bispecific antibodies include those described in WO2022 / 059766 and shown in Table 1 below. Preferred examples include the bispecific antibodies shown in (1) to (14) in Table 1 below. These antibodies are bispecific antibodies that bind to a complex formed by HLA-DQ2.5 and gluten peptides.

[0289] [Table 1] --------------------------------------------------- (1) DQN0344H0976 / L0591 / / DQN0385H1270 / L0722-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1270 / L0722) (2) DQN0344H0976 / L0591 / / DQN0385H1270 / L0681-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1270 / L0681) (3) DQN0344H0976 / L0591 / / DQN0385H1352 / L0681-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1352 / L0681) (4) DQN0344H0976 / L0591 / / DQN0385H1527 / L0605-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1527 / L0605) (5) DQN0344H0976 / L0591 / / DQN0385H1255 / L0605-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1255 / L0605) (6) DQN0344H1013 / L0620 / / DQN0385H1270 / L0722-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1270 / L0722) (7) DQN0344H1013 / L0620 / / DQN0385H1521 / L0605-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1521 / L0605) (8) DQN0344H1013 / L0620 / / DQN0385H1270 / L0681-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1270 / L0681) (9) DQN0344H1013 / L0620 / / DQN0385H1352 / L0681-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1352 / L0681) (10) DQN0344H1013 / L0620 / / DQN0385H1353 / L0681-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1353 / L0681) (11)DQN0344H0976 / L0591 / / DQN0385H1521 / L0605-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1521 / L0605) (12) DQN0344H0976 / L0591 / / DQN0385H1353 / L0681-F6 (Bispecific antibody of DQN0344H0976 / L0591 and DQN0385H1353 / L0681) (13) DQN0344H1013 / L0620 / / DQN0385H1255 / L0605-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1255 / L0605) (14) DQN0344H1013 / L0620 / / DQN0385H1527 / L0605-F6 (Bispecific antibody of DQN0344H1013 / L0620 and DQN0385H1527 / L0605) (15) A first polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the first heavy chain sequence of the bispecific antibody of any one of (1) to (14); a second polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the first light chain sequence of the bispecific antibody of any one of (1) to (14); any one of (1) to (14). a third polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the second heavy chain sequence of the bispecific antibody of (1); and a fourth polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the second light chain sequence of the bispecific antibody of any one of (1) to (14).

[0290] More specifically, examples of such bispecific antibodies include those comprising a combination of four polypeptide chains selected from the group consisting of (1) to (15) in Table 2 below. Note that (1) to (14) in Table 2 correspond to (1) to (14) in Table 1, and the antibodies in (1) to (14) in Table 2 are antibodies having the heavy chain / light chain combinations shown in (1) to (14) in Table 1, respectively. Preferred examples of bispecific antibodies used in the present invention include the bispecific antibodies shown in (1) to (14) in Table 2 below. These antibodies are bispecific antibodies that bind to a complex formed by HLA-DQ2.5 and gluten peptides.

[0291] [Table 2] --------------------------------------------------- (1) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second light chain comprising the amino acid sequence of SEQ ID NO: 6; (2) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (3) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 8 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (4) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; (5) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; (6) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second light chain comprising the amino acid sequence of SEQ ID NO: 6; (7) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; (8) (9) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (10) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 8 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7;(10) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (11) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; (12) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a second light chain comprising the amino acid sequence of SEQ ID NO: 7; (13) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; (14) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a first light chain comprising the amino acid sequence of SEQ ID NO: 4, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a second light chain comprising the amino acid sequence of SEQ ID NO: 10; and (15) A first polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a first heavy chain sequence described in any one of (1) to (14); a second polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a first light chain sequence described in any one of (1) to (14); a third polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a second heavy chain sequence described in any one of (1) to (14); and a fourth polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a second light chain sequence described in any one of (1) to (14). ──────────────────────────────────;

[0292] Furthermore, the bispecific antibody described in WO2022 / 059766 above is a bispecific antibody comprising a combination of four polypeptide chains selected from the group consisting of (1) and (2) below. Particularly preferred bispecific antibodies of the present invention are bispecific antibodies comprising a combination of four polypeptide chains described in (1). These antibodies are bispecific antibodies that bind to a complex formed by HLA-DQ2.5 and gluten peptides. (1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a first light chain comprising the amino acid sequence of SEQ ID NO: 15, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a second light chain comprising the amino acid sequence of SEQ ID NO: 17; or (2) a first polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the first heavy chain sequence described in (1); a second polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the first light chain sequence described in (1); a third polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the second heavy chain sequence described in (1); and a fourth polypeptide chain consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the second light chain sequence described in (1).

[0293] Antibodies include not only monoclonal antibodies derived from animals such as humans, mice, rats, hamsters, rabbits, and monkeys, but also artificially modified recombinant antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies. Recombinant antibodies may be chemically modified, for example, by conjugation with various molecules such as polyethylene glycol. Furthermore, the class of the antibody is not particularly limited, and the immunoglobulin class of the antibody is not particularly limited, and may be any class, such as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA, IgD, IgE, or IgM, with IgG and IgM being preferred. IgG1 and IgG4 are particularly preferred, with IgG1 being particularly preferred for application of the present invention.

[0294] In the present invention, examples of antibody fragments include antigen-binding fragments of antibodies. Antigen-binding fragments of antibodies preferably include fragments containing two heavy chains and two light chains, and F(ab')2 is a preferred example of such fragments. The present invention is also useful for producing bivalent antibodies in which two Fabs are artificially linked via a disulfide bond or a non-disulfide bond, even if the antibody does not have a disulfide bond in the hinge region like natural F(ab')2.

[0295] As already mentioned, the antibodies or fragments thereof of the present invention include those comprising two heavy chains and two light chains. Specifically, the antibodies include those comprising two heavy chains that are different from each other (i.e., heavy chains with different amino acid sequences) and two light chains that are different from each other (i.e., light chains with different amino acid sequences). Such antibodies comprise heterogeneous heavy chains and heterogeneous light chains. Furthermore, the epitope bound by a first heavy chain and light chain pair and the epitope bound by a second heavy chain and light chain pair may be the same or different, but are preferably different. Furthermore, the target molecule bound by a first heavy chain and light chain pair and the target molecule bound by a second heavy chain and light chain pair may be the same molecule or different molecules. In a preferred embodiment, the antibody is a multispecific antibody, for example, a bispecific antibody.

[0296] Furthermore, the present invention is preferably applicable to heavy chains that are different from each other and that are more prone to heteropairing than homopairing, and specifically, one or both of the two heavy chains may have a mutation that promotes heteropairing and / or suppresses homopairing. Examples of such a mutation include a mutation in at least one of the two heavy chains that suppresses homopairing of that chain due to steric hindrance, and an example of such a mutation is known as a knob-into-hole.

[0297] Furthermore, at least one of the two heavy chains may have a mutation that suppresses homopairing of that chain due to charge repulsion. Specifically, at least one of the two heavy chains may have a charged amino acid that suppresses homopairing of that chain. For example, one of the two heavy chains may have a positively or negatively charged amino acid introduced into it, and the charged amino acids may repel each other, thereby suppressing homopairing of the heavy chain. Alternatively, one of the two heavy chains may have a positively or negatively charged amino acid introduced into it, and an oppositely charged amino acid introduced into the other chain. For example, a positively or negatively charged amino acid introduced into one of the two heavy chains may attract an oppositely charged amino acid introduced into the other chain, thereby promoting heteropairing of the heavy chain.

[0298] The present invention is also suitable for antibodies having two different heavy chains and two different light chains, in which pairing between each heavy chain and its corresponding light chain is more likely than pairing between an unmatched light chain. For example, pairing between a heavy chain and its corresponding light chain may be more likely than pairing between an unmatched light chain due to electrical attraction between positively charged amino acids and negatively charged amino acids, or pairing between a heavy chain and its unmatched light chain may be more difficult than pairing between an unmatched light chain due to electrical repulsion between like-charged amino acids.

[0299] Specifically, the two heavy chains may be different from each other, the two light chains may also be different from each other, and at least one of the two heavy chains and the two light chains may have a mutation introduced into it that promotes pairing of the corresponding heavy chain / light chain and / or suppresses pairing of a non-corresponding heavy chain / light chain. Alternatively, the two heavy chains may be different from each other, the two light chains may also be different from each other, and at least one heavy chain and its corresponding light chain may have a mutation introduced into it that promotes pairing between that heavy chain and light chain. Alternatively, the two heavy chains may be different from each other, the two light chains may also be different from each other, and at least one heavy chain and its corresponding light chain may have a mutation introduced into it that suppresses pairing between that heavy chain and light chain. Alternatively, the two heavy chains may be different from each other, and the two light chains may be different from each other, and mutations that promote pairing between corresponding heavy and light chains and / or suppress pairing between non-corresponding heavy and light chains may be introduced into all of the two heavy and light chains. Such mutations include, for example, mutations that introduce charged amino acids as described herein.

[0300] For example, antibodies can be mentioned in which a positively or negatively charged amino acid is introduced into at least one of the two heavy chains, and an oppositely charged amino acid is introduced into the corresponding light chain, and / or an identically charged amino acid is introduced into the non-corresponding light chain. Specifically, the electrically charged amino acid in the heavy chain into which the positively or negatively charged amino acid has been introduced and the oppositely charged amino acid introduced into the corresponding light chain are electrically attracted to each other, and / or the electrically charged amino acid in the non-corresponding light chain is electrically repulsed, thereby promoting pairing between the heavy chain and the corresponding light chain and / or suppressing pairing between the heavy chain and the non-corresponding light chain, respectively. For details of these antibodies, please refer to the descriptions herein and known techniques.

[0301] The obtained antibody or fragment thereof can be purified to homogeneity. Separation and purification of antibodies or fragments thereof can be performed using methods commonly used for polypeptides. For example, antibodies can be separated and purified by appropriately selecting and combining methods such as high-performance liquid chromatography, ion exchange chromatography, size exclusion chromatography, and affinity chromatography, as well as filters, ultrafiltration, salting out, dialysis, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). The concentration of the antibody obtained above can be measured by absorbance measurement or enzyme-linked immunosorbent assay (ELISA), etc.

[0302] Columns used for affinity chromatography include protein A columns and protein G columns. For example, columns using protein A columns include Hyper D, POROS, and Sepharose FF (Pharmacia).

[0303] Examples of chromatography other than affinity chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographies can be performed using liquid-phase chromatography such as HPLC and FPLC.

[0304] Before or after purification, the polypeptide can be treated with an appropriate polypeptide-modifying enzyme, such as trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, or glucosidase, to optionally modify or partially remove peptides.

[0305] If the antibody or fragment thereof produced by the method of the present invention has biological activity that allows it to be used as a pharmaceutical, a drug can be produced by mixing the polypeptide with a pharmaceutically acceptable carrier or additive and formulating it (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). The present invention relates to compositions, particularly pharmaceutical compositions, pharmaceutical preparations, and pharmaceutical formulations, comprising the antibody or fragment thereof produced by the method of the present invention and a pharmaceutically acceptable carrier.

[0306] Examples of pharmaceutically acceptable carriers and additives include water, buffer solutions, excipients, stabilizers, preservatives, and the like, such as pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, and the like.

[0307] The actual additives may be selected from the above, either singly or in combination, depending on the dosage form of the therapeutic agent of the present invention, but are not limited thereto. For example, when used as an injectable formulation, the purified polypeptide may be dissolved in a solvent such as physiological saline, buffer solution, or glucose solution, to which an anti-adsorption agent such as Tween® 80, Tween® 20, gelatin, or human serum albumin may be added. Alternatively, the polypeptide may be lyophilized to prepare a dosage form that is dissolved and reconstituted before use. For example, sugar alcohols or saccharides such as mannitol and glucose may be used as excipients for lyophilization. Exemplary lyophilized formulations are described in U.S. Patent No. 6,267,958. Aqueous solution formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0308] The effective dose of an antibody or a fragment thereof is selected as appropriate depending on the type of antibody or fragment thereof, the type of disease to be treated or prevented, the age of the patient, the severity of the disease, and the like. For example, the effective dose of an antibody is selected from the range of 0.001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dose per patient can be selected from the range of 0.01 to 100,000 mg / body. However, the dose is not limited to these doses.

[0309] The antibody or a fragment thereof can be administered orally or parenterally, but is preferably administered parenterally. Specific examples include injection (for example, systemic or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.), nasal administration, pulmonary administration, and transdermal administration.

[0310] All documents cited herein are hereby incorporated by reference.

[0311] The following are examples of the methods and compositions of the present invention. These are provided to aid in understanding the invention, but should not be construed as limiting the scope of the invention. It will be understood that various other embodiments may be practiced in light of the general description above.

[0312] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and illustrations herein should not be construed as limiting the scope of the invention.

[0313] Example 1: Construction of expression vectors for bispecific antibodies to HLA-DQ2.5 / gluten peptide complexes The 14 bispecific antibodies listed in (1) to (14) of Table 1 (specifically, the 14 bispecific antibodies consisting of the heavy and light chain combinations listed in (1) to (14) of Table 2) were created using FAST-Ig® technology and are humanized IgG1 bispecific antibodies that bind to the complex formed by HLA-DQ2.5 and gluten peptides. From these 14 bispecific antibodies, three representative bispecific antibodies were selected, specifically antibodies (5), (7), and (8) of Table 1, and more specifically bispecific antibodies (5), (7), and (8) of Table 2, respectively, and named 3CC1, 3CC2, and 3CC3, respectively. For each bispecific antibody, a CAG promoter was attached upstream of the genes for the first heavy chain (H1 chain), second heavy chain (H2 chain), first light chain (L1 chain) corresponding to the first heavy chain, and second light chain (L2 chain) corresponding to the second heavy chain, and a rabbit β-globin polyA signal sequence was attached downstream to construct an antibody gene expression unit. All three selected bispecific antibodies have a first heavy chain (H1 chain) consisting of a polypeptide with the amino acid sequence of SEQ ID NO: 14, a first light chain (L1 chain) corresponding to the first heavy chain consisting of a polypeptide with the amino acid sequence of SEQ ID NO: 15, a second heavy chain (H2 chain) consisting of a polypeptide with the amino acid sequence of SEQ ID NO: 16, and a second light chain (L2 chain) corresponding to the second heavy chain consisting of a polypeptide with the amino acid sequence of SEQ ID NO: 17.

[0314] Specifically, 3CC1 has a first heavy chain (H1 chain) consisting of a polypeptide with the amino acid sequence of SEQ ID NO: 1, a first light chain (L1 chain) corresponding to the first heavy chain consisting of a polypeptide with the amino acid sequence of SEQ ID NO: 2, a second heavy chain (H2 chain) consisting of a polypep...

Claims

1. A recombinant vector comprising four coding regions in a 1:1:1:1 ratio, each coding region encoding two different antibody heavy chains or antigen-binding fragments thereof and two different light chains or antigen-binding fragments thereof corresponding to each heavy chain.

2. The vector of claim 1, wherein the antibody is a multispecific antibody.

3. The vector of claim 1, wherein the pairing of one heavy chain of the antibody with itself and the pairing of the other heavy chain with itself are inhibited by steric hindrance due to knob-into-hole and / or charge repulsion of charged amino acids.

4. The vector of claim 1, wherein pairing of one heavy chain of the antibody with the light chain corresponding to the other heavy chain, and pairing of the other heavy chain with the light chain corresponding to the one heavy chain, are inhibited by charge repulsion of charged amino acids.

5. The vector of claim 1, wherein the antibody is an antibody that binds to a complex formed by HLA-DQ2.5 and a gluten peptide.

6. The vector of claim 1, which contains two copies of each of the four coding regions.

7. The vector of claim 1, wherein the four coding regions are each transcribed as a different mRNA molecule.

8. The vector of claim 7, wherein each of the four coding regions is linked to a promoter.

9. The vector of claim 1, which is a mammalian expression vector.

10. A cell into which the vector described in claim 1 has been introduced.

11. The cell of claim 10, wherein the vector is introduced into the genome of the cell.

12. The cell of claim 10, wherein the vector is introduced into the genome in two copies.

13. The cell according to claim 10, which contains four copies of each of the four coding regions on its genome.

14. The cell of claim 10, which is a mammalian cell.

15. A method for producing a molecule, including an antibody or antigen-binding fragment, comprising expressing the vector in the cell of claim 10.

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